JP2020532965A - Anti-CD137 molecule and its use - Google Patents

Abstract

The present disclosure discloses an antibody that binds to human CD137 or an antigen-binding fragment thereof, a nucleic acid that encodes the antibody, a therapeutic composition thereof, and T cell function is enhanced to upward control the cell-mediated immune response and T such as tumor immunity. Provided are their use for treating cell-mediated dysfunction and for treating cancer. [Selection diagram] None

Description

Cross-references to related applications This application claims the preferred interests of international patent application serial number PCT / CN2017 / 0983332 filed on August 21, 2017, which is incorporated herein by reference in its entirety.

References to the Sequence Listing The contents of the following submissions regarding ASCII text files are incorporated herein by reference in their entirety: Computer-readable format (CRF) of the sequence listing (filename: 695402000341secrist.txt, date of recording: 2018). August 20, size: 542KB).

The present disclosure relates to the use of antibodies that bind to human CD137 or antigen-binding fragments thereof, nucleic acids encoding them, therapeutic compositions thereof, and their antitumors.

CD137 (also called CD137 receptor, 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). The current understanding of CD137 is that its expression is generally activation-dependent, activated NK and NKT cells, regulatory T cells, dendritic cells (DCs), stimulating mast cells, differentiated bone marrow cells, monocytes, eosinophils. It has been shown to be present in a broad subset of immune cells, including monocytes and eosinophils (Wang, 2009, Immunological Reviews 229: 192-215). Expression of CD137 is also associated with the tumor vasculature (Broll, 20011, Amer. J. Clin. Pathol. 115 (4): 543-549; Seaman, 2007, Cancer Cell 11: 538-554) and inflammatory or atherosclerosis. It has also been demonstrated at the site of the endothelium of (Drencard, 2007 FASEB J. 21: 456-436; Olofsson, 2008, Inflammation 117: 1292-1301). The ligand that stimulates CD137, the CD137 ligand (CD137L), is expressed in activated antigen presenting cells (APCs), myeloid progenitor cells, and hematopoietic stem cells.

Human CD137 is a protein of 255 amino acids (GenBank Accession No. NM _{_} 001561; NP _{_} 001552; SEQ ID NO: 1). The protein comprises a signal sequence (amino acid residues 1-17) followed by an extracellular domain (169 amino acids), a transmembrane domain (27 amino acids), and an intracellular domain (42 amino acids) (Cheek ATC et al. 2004 Cancer). Domain Protein 11: 215-226). Receptors can be expressed on the cell surface in the form of monomers and dimers, trimerized with CD137 ligands and signaled.

Numerous studies of mouse and human T cells have shown that CD137 promotes increased cell proliferation, survival, and cytokine production (Croft, 2009, Nat Rev Immunol 9: 271-285). Studies have shown that several CD137 agonists, mAbs, increase the expression of costimulatory molecules, significantly enhance the cytolytic T-lymphocyte response, and provide antitumor effects in various models. The CD137 agonist mAb has demonstrated efficacy in prophylactic and therapeutic environments. In addition, tumor models of CD137 monotherapy and combination therapy have established a permanent antitumor protective T cell memory response (Lynch, 2008, Immunol Rev. 22: 277-286). CD137 agonists have also been shown to suppress autoimmune responses in a variety of autoimmune models recognized in the art (Viney, 2006, J Mol Med 84: 726-736). This two-sided activity of CD137 presents the potential to provide antitumor activity while suppressing autoimmune side effects that may be associated with immunotherapeutic approaches that break immune tolerance.

The need for antibodies that bind to human CD137 and increase the response via CD137, thereby providing promising therapies for the treatment of various diseases and conditions, including cancer and autoimmune diseases, remains unmet. In addition, there is a need for anti-CD137 antibodies that cross-react between different species such as humans and laboratory animals (mice, monkeys, dogs, etc.) in order to enable animal model studies and at the same time provide treatment candidates.

An object of the present disclosure is to provide an isolated binding molecule that binds to human CD137, such as an antibody or binding fragment thereof, or a derivative thereof. Another object of the present disclosure is to provide a composition comprising a binding molecule that binds to CD137. It is also an object of the present disclosure to provide a method of treating a disease and / or condition associated with or mediated by CD137 signaling by using one or more binding molecules of the present disclosure. These and other purposes of the present disclosure are described more fully herein.

Thus, in one aspect, provided herein is one or more (eg, two or more, three or more, four or more) of the following functional properties that bind to the extracellular domain of human CD137. One or more antibodies (eg, isolated antibodies), or one or more of them, including 5, or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10). It is an antigen-binding fragment: (a) binds to one or more amino acid residues in amino acid residues 34-108 of SEQ ID NO: 1; (b) amino acid residues 109-112, 125, 126 of SEQ ID NO: 1. does not bind to one or more amino acid residues 135～138,150 and 151; binds to human CD137 with (c) 100 nM following _{K D;} (d) have agonist activity against human CD137; (e) 1000 nM Does not bind to human OX40, CD40, GITR, and / or CD27 receptors at concentrations up to; (f) cross-reactive with monkey, mouse, rat, and / or canine CD137; (g) ADCC effect. It does not induce; (h) it can inhibit tumor cell growth; (i) it has a therapeutic effect on cancer; and / or (j) it blocks the binding between CD137 and CD137L.

Thus, in one aspect, provided herein is an antibody that binds to the extracellular domain of human CD137 (eg, an isolated antibody) or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, where HVR-H1 Includes an amino acid sequence according to a formula selected from the group consisting of: formula (I): X ₁ TFX ₂ X ₃ YX ₄ IHWV (SEQ ID NO: 2), in which X1 is F or Y and X2 is S. Or T, X3 is G, N, or S, and X4 is A, G, or W; formula (II): YSIX ₁ SGX ₂ X ₃ WX ₄ WI (SEQ ID NO: 3), formula. Among them, X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and formula (III) :. FSLSTX ₁ GVX ₂ VX ₃ WI (SEQ ID NO: 4), in the formula, X1 is G or S, X2 is A or G, and X3 is A, G, S, or T; HVR-H2 is , Containing an amino acid sequence according to a formula selected from the group consisting of: formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO: 5), in which X1 is A, D or Y and X2 is D or G X3 is R, S, or Y, and X4 is P or T; formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO: 6), in the formula, X1 is D or E and X2 is N or S. , And X3 are N or S; and formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 7), in the formula, X1 is A, G, S, V, or Y, and X2 is A, D, S, or Y, X3 is D, G, or S, X4 is S or T; and HVR-H3 comprises the amino acid sequence according to formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO: 8), in formula X1 Is E or G, X2 is E or S, X3 is D or T, X4 is A, T, or V, X5 is A, I, L, T, or V, and X6 is A, D, or G.

In some embodiments, provided herein is an antibody (eg, an isolated antibody) that binds to the extracellular domain of human CD137, including heavy and light chain variable regions, or an antibody thereof. An antigen-binding fragment, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, where HVR-H1 comprises an amino acid sequence according to a formula selected from the following group: formula (XII). : X1TFSX2YWIHWV (SEQ ID NO: 853), in formula X1 is F or Y, and X2 is N or S; formula (XIII): YSIX1SGX2X3WX4WI (SEQ ID NO: 854), in formula X1 is S or T , X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, or S; and formula (XIV): FSLSTX1GVX2VX3WI (SEQ ID NO: 855), in formula X1 Is G or S, X2 is A or G, and X3 is A, G, or S; HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (IV). ): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO: 5), in the formula, X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T. And the formula (XV): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 856), in the formula, X1 is G, S, V, or Y, X2 is A, D, S, or Y, and X3 is D, G, or S, X4 is S or T; and HVR-H3 comprises the amino acid sequence according to formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO: 8), where X1 is E or G and X2 is E or S, X3 is D or T, X4 is A, T, or V, X5 is A, I, L, T, or V, and X6 is A, D, or G.

In another aspect, provided herein is an antibody that binds to the extracellular domain of human CD137 (eg, an isolated antibody) or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, where HVR-L1 , Including the amino acid sequence according to formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO: 9), in the formula, where X1 is Q or R, X2 is D, G, or S, X3 is I or V, and X4 is. G, R, S, or T, X5 is P, R, S, or T, X6 is A, D, F, S, V, or Y, X7 is L or V, and X8 is A, G, or N; HVR-L2 comprises the amino acid sequence according to formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO: 10), where X1 is A or D and X2 is N, S, Or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and HVR-L3 consists of: Including the amino acid sequence according to the formula selected from the group: formula (X): YCQQX1YX2X3X4T (SEQ ID NO: 11), in the formula, X1 is A, G, S, or Y and X2 is Q, S, or Y. , X3 is I, L, T, or Y, and X4 is I, S, V, or W; and formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO: 12), where X1 is E or Q. Yes, X2 is P, S, or Y, X3 is D, L, S, T, or Y, X4 is D, E, H, S, or T, and X5 is D, L, T. , Or W, and X6 is L, P, R, or V.

In some embodiments, provided herein is an antibody (eg, an isolated antibody) that binds to the extracellular domain of human CD137, including heavy and light chain variable regions, or an antibody thereof. An antigen binding fragment, the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, which comprises the amino acid sequence according to formula (XVI): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO: 857), in the formula. , X1 is Q or R, X2 is D, G, or S, X3 is I or V, X4 is G, R, S, or T, and X5 is P, R, S, or T, X6 is A, F, S, V, or Y, X7 is L or V, and X8 is A or G; HVR-L2 comprises the amino acid sequence according to formula (XVII). : X ₁ ASX ₂ X ₃ X ₄ X ₅ GX ₆ (SEQ ID NO: 858), in the formula, X1 is A or D, X2 is N or S, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and HVR-L3 comprises the amino acid sequence according to formula (XVIII): YCQQX ₁ YX ₂ X ₃ WT (SEQ ID NO: 859), in the formula, X1 is A or G, X2 is S or Y, and X3 is I, L, or T.

In another aspect, provided herein is an antibody that binds to the extracellular domain of human CD137 (eg, an isolated antibody) or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable regions being HVR-H1, HVR-H2, and HVR-H3, which are: Including: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH VH25, VH26, VH27, VH28, VH29, VH30, VH31, VH32, VH33, VH34, VH35, VH36, VH37, VH38, VH39, VH40, VH41, VH42, VH43, VH44, VH45, VH46, VH47, VH48 VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58, VH59, or VH60; and / or the light chain variable regions are HVR-L1, HVR-L2, and HVR-L3, which are: Includes: VL1, VL2, VH3, VL4, VH5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, , VL25, VL26, VL27, VL28, VL29, VL30, VL31, VL32, VL33, VL34, VL35, VL36, VL37, VL38, VL39, VL40, VL41, VL42, VL43, VL44, VL45, VL46, VL47, VL48 , VL50, VL51, VL52, VL53, VL54, VL55, VL56, VL57, VL58, VL59, or VL60 (shown in Table 1c). In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are as follows: HVR-H1, HVR-H2, Includes HVR-H3, HVR-L1, HVR-L2, and HVR-L3: VH1 and VL1, VH2 and VL2, VH3 and VL3, VH4 and VL4, VH5 and VL5, VH6 and VL6, VH7 and VL7, VH8 and VL8. , VH9 and VL9, VH10 and VL10, VH11 and VL11, VH12 and VL12, VH13 and VL13, VH14 and VL14, VH15 and VL15, VH16 and VL16, VH17 and VL17, VH18 and VL18, VH19 and VL19, VH20 and VL20. And VL21, VH22 and VL22, VH23 and VL23, VH24 and VL24, VH25 and VL25, VH26 and VL26, VH27 and VL27, VH28 and VL28, VH29 and VL29, VH30 and VL30, VH31 and VL31, VH32 and VL32, VL32. , VH34 and VL34, VH35 and VL35, VH36 and VL36, VH37 and VL37, VH38 and VL38, VH39 and VL39, VH40 and VL40, VH41 and VL41, VH42 and VL42, VH43 and VL43, VH44 and VL44, VH45 and VL45. And VL46, VH47 and VL47, VH48 and VL48, VH49 and VL49, VH50 and VL50, VH51 and VL51, VH52 and VL52, VH53 and VL53, VH54 and VL54, VH55 and VL55, VH56 and VL56, VH57 and VL57, VL58. , VH59 and VL59, or VH60 and VL60 (shown in Table 1c).

In another aspect, provided herein is an antibody that binds to the extracellular domain of human CD137 (eg, an isolated antibody) or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain variable region which is: VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH26, VH27 VH29, VH30, VH31, VH32, VH33, VH34, VH35, VH36, VH37, VH38, VH39, VH40, VH41, VH42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52 VH54, VH55, VH56, VH57, VH58, VH59, or VH60; and / or the light chain variable regions include: VL1, VL2, VH3, VL4, VH5, VL6, VL7. , VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, VL16, VL17, VL18, VL19, VL20, VL21, VL22, VL23, VL24, VL25, VL26, VL27, VL28, VL29, VL30, VL31 , VL33, VL34, VL35, VL36, VL37, VL38, VL39, VL40, VL41, VL42, VL43, VL44, VL45, VL46, VL47, VL48, VL49, VL50, VL51, VL52, VL53, VL54, VL55, VL56 , VL58, VL59, or VL60 (shown in Table 1c). In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are the following: heavy chain variable region and light chain variable region. Includes regions: VH1 and VL1, VH2 and VL2, VH3 and VL3, VH4 and VL4, VH5 and VL5, VH6 and VL6, VH7 and VL7, VH8 and VL8, VH9 and VL9, VH10 and VL10, VH11 and VL11, VH12 and VL12, VH13 and VL13, VH14 and VL14, VH15 and VL15, VH16 and VL16, VH17 and VL17, VH18 and VL18, VH19 and VL19, VH20 and VL20, VH21 and VL21, VH22 and VL22, VH23 and VL23, VH24 VH25 and VL25, VH26 and VL26, VH27 and VL27, VH28 and VL28, VH29 and VL29, VH30 and VL30, VH31 and VL31, VH32 and VL32, VH33 and VL33, VH34 and VL34, VH35 and VL35, VH36 and VL36. VL37, VH38 and VL38, VH39 and VL39, VH40 and VL40, VH41 and VL41, VH42 and VL42, VH43 and VL43, VH44 and VL44, VH45 and VL45, VH46 and VL46, VH47 and VL47, VH48 and VL48, VH49 VH50 and VL50, VH51 and VL51, VH52 and VL52, VH53 and VL53, VH54 and VL54, VH55 and VL55, VH56 and VL56, VH57 and VL57, VH58 and VL58, VH59 and VL59, or VH60 and VL60 (Table 1c). ).

In another aspect, provided herein is an antibody that binds to the extracellular domain of human CD137 (eg, an isolated antibody) or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment binds to one or more amino acid residues within amino acid residues 34-93 of SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO: 1. .. In some embodiments, the antibody or antigen binding fragment of SEQ ID NO: 1 is one or more of amino acid residues 34-36, one or more of 53-55, and one of 92-93. Combine one or more. In some embodiments, the antibody or antigen binding fragment is among the amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. Do not combine into one or more. In some embodiments, the antibody or antigen binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment is cross-reactive with a CD137 polypeptide from at least one non-human species selected from cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, the antibody or antigen binding fragment binds to cynomolgus monkey CD137.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, where the heavy chain variable region is HVR-H1, comprising the amino acid sequence of SEQ ID NO: 711, the amino acid sequence of SEQ ID NO: 735. Contains HVR-H2 comprising, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 759, and / or the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 783, the amino acid sequence of SEQ ID NO: 807. Includes HVR-L2 and HVR-L3 containing the amino acid sequence of SEQ ID NO: 831. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 617, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 618.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, where the heavy chain variable region is HVR-H1, comprising the amino acid sequence of SEQ ID NO: 712, the amino acid sequence of SEQ ID NO: 736. Contains HVR-H2 comprising, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 760, and / or the light chain variable region comprises HVR-L1, comprising the amino acid sequence of SEQ ID NO: 784, the amino acid sequence of SEQ ID NO: 808. Includes HVR-L2 and HVR-L3 containing the amino acid sequence of SEQ ID NO: 832. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 619, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 620.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, where the heavy chain variable region is HVR-H1, comprising the amino acid sequence of SEQ ID NO: 731, the amino acid sequence of SEQ ID NO: 755. Contains HVR-H2 comprising, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 779, and / or the light chain variable region comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 803, the amino acid sequence of SEQ ID NO: 827. Includes HVR-L2 and HVR-L3 containing the amino acid sequence of SEQ ID NO: 851. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 657, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 658.

In another aspect, provided herein is an amino acid (eg, an isolated amino acid) that binds to the extracellular domain of human CD137, comprising a heavy chain variable region and a light chain variable region, and is heavy. The chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, and HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: formulas (I), (II), and Formula (III); HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (IV), formula (V), and formula (VI); HVR-H3 according to formula (VII). Containing the amino acid sequence; and / or the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, HVR-L1 comprises the amino acid sequence according to formula (VIII); HVR-L2 contains formula (IX). Contains the amino acid sequence according to; HVR-L3 contains the amino acid sequence according to the formula selected from the group consisting of: formulas (X) and formula (XI). In some embodiments, provided herein are an amino acid (eg, an isolated amino acid) that binds to the extracellular domain of human CD137, including heavy and light chain variable regions. The heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, and HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (XIII) and formula (XVI). HVR-H2 contains an amino acid sequence according to a formula selected from the group consisting of: formula (IV) and formula (XV); HVR-H3 contains an amino acid sequence according to formula (VII); and / or a light chain. The variable region comprises HVR-L1, HVR-L2, and HVR-L3, HVR-L1 contains an amino acid sequence according to formula (XVI); HVR-L2 contains an amino acid sequence according to formula (XVII); HVR-L3 contains an amino acid sequence according to formula (XVII). Contains the amino acid sequence according to formula (XVIII).

In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 253 to 312, and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 313 to 372. , HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 373 to 432, HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 492, and HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 492. It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 493 to 552, and HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 553 to 612. In some embodiments, the heavy chain variable region is SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47. , 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97 , 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, and / or the amino acid sequence selected from the group consisting of 131. The light chain variable regions are SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, Includes an amino acid sequence selected from the group consisting of 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, and 132.

In some embodiments, HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of formula (XII), formula (XIII), and formula (XIV); HVR-H21 comprises formula (IV). Or contains an amino acid sequence according to formula (XV); and HVR-H3 contains an amino acid sequence according to formula (VII); and / or HVR-L1 contains an amino acid sequence according to formula (XVI); HVR-L2 contains an amino acid sequence according to formula (XVI). Contains the amino acid sequence according to XVII); HVR-L3 contains the amino acid sequence according to formula (XVIII). In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 709-732 and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 733-756. , HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 757 to 780, HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 781 to 804, and HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 781 to 804. It comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 805-828, and HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 829-852. In some embodiments, the heavy chain variable region is SEQ ID NO: 15, 17, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 61, 63, 65, 67, 71. , 73, 75, 79, 83, 85, and 87, and / or the light chain variable region comprises SEQ ID NOs: 16, 18, 32, 34, 36, 38, 40, Includes an amino acid sequence selected from the group consisting of 42, 44, 46, 48, 50, 54, 62, 64, 66, 68, 72, 74, 76, 80, 84, 86, and 88. In some embodiments, the antibody comprises heavy and light chains, the heavy chains being SEQ ID NOs: 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639. , 641, 643, 645, 647, 649, 651, 653, 655, 657, and 659, and / or the light chain comprises SEQ ID NOs: 614, 616, 618, 620. , 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, and 660. including.

In some embodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 711 or 731, HVR-H2 comprises the amino acid sequence of SEQ ID NO: 735 or 755, and HVR-H3 comprises the amino acid sequence of SEQ ID NO: 759 or 779. HVR-L1 comprises the amino acid sequence of SEQ ID NO: 783 or 803, HVR-L2 comprises the amino acid sequence of SEQ ID NO: 807 or 827, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 831 or 851. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 or 71, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42 or 72. In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 617 or 657, and the light chain comprises the amino acid sequence of SEQ ID NO: 618 or 658.

In some embodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 712, HVR-H2 comprises the amino acid sequence of SEQ ID NO: 736, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 760, and HVR-L1 Contains the amino acid sequence of SEQ ID NO: 784, HVR-L2 contains the amino acid sequence of SEQ ID NO: 808, and HVR-L3 contains the amino acid sequence of SEQ ID NO: 832. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 619, and the light chain comprises the amino acid sequence of SEQ ID NO: 620.

In some embodiments that may be combined with any of the above embodiments, an antibody or antigen-binding fragments (for example, when measured by surface plasmon resonance) binds to human CD137 with 100nM following K _D. In some embodiments, the antibody or antigen-binding fragments (for example, when measured by surface plasmon resonance) binds to human CD137 with 50nM following K _D.

In some embodiments that can be combined with any of the aforementioned embodiments, the antibody or antigen binding fragment is a crab monkey (eg, GenBank gene ID 102127961), a mouse (eg, GenBank gene ID 21942), a rat (eg, GenBank gene ID 500590). ) And / or cross-reactivity with a CD137 polypeptide from at least one non-human species selected from dogs (eg, GenBank gene ID 608274). In some embodiments, the antibody or antigen binding fragment binds to cynomolgus monkey CD137.

In some embodiments that can be combined with any of the aforementioned embodiments, contact with an antibody or antigen-binding fragment reduces the activity of human CD137 (eg, when expressed in cells such as human cells).

In some embodiments that can be combined with any of the aforementioned embodiments, the antibody or antigen binding fragment has a half inhibitory concentration of about 100 nM or less to block the binding of human CD137 to human CD137L in vitro. It has an IC ₅₀ ). In some embodiments, the antibody or antigen-binding fragment completely blocks the binding of human CD137 to human CD137L in vitro when the antibody or antigen-binding fragment is provided at a concentration of about 1 μM or higher. In some embodiments that can be combined with any of the aforementioned embodiments, contact with an antibody or antigen-binding fragment increases the activity of human CD137 (eg, when expressed in cells such as human cells). In some embodiments, contacting CD137 (eg, expressed in human cells) with an antibody or antigen-binding fragment increases NF-κB-dependent transcription. In some of the embodiments described above, the antibody or antigen binding fragment is stimulated in one or more embodiments of CD137 signaling stimulated by CD137L, such as CD137L, in cells expressing CD137. Blocks NF-κB-dependent transcription.

In some embodiments that can be combined with any of the aforementioned embodiments, the antibody comprises a human IgG2 Fc region. In some embodiments that can be combined with any of the aforementioned embodiments, the antibody comprises a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises the S241P mutation, where numbering is by Kabat. In some embodiments, the antibody or antigen binding fragment does not induce ADCC effect.

In another aspect, provided herein are SEQ ID NOs: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165. , 167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215 , 217, 219, 221 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251 encoded by a polynucleotide containing a sequence selected from Antibody heavy chain variable region and / or SEQ ID NO: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, Antibody light chain variable region encoded by a polynucleotide comprising a sequence selected from 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252. Is. In some embodiments, the references provided herein are SEQ ID NOs: 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691. , 693, 695, 697, 699, 701, 703, 705, and antibody heavy chains encoded by polynucleotides containing sequences selected from 707, and / or SEQ ID NOs: 662, 664, 666, 668, 670, Encoded into a polynucleotide containing a sequence selected from 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708. It is an antibody light chain.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1 containing the sequence GFSLSTGVGVG (SEQ ID NO: 866), HVR-H2 containing the sequence LIDWDDDKYSPSLKS (SEQ ID NO: 867), and HVR-H3 containing the sequence GGSDTLVDWFAY (SEQ ID NO: 868). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQSVSPYLA (SEQ ID NO: 869), HVR-L2 containing the sequence DASSLES (SEQ ID NO: 870), and HVR containing the sequence QQGYSLWT (SEQ ID NO: 871). Includes a light chain variable (VH) domain containing −L3.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1, which comprises the sequence GYSITSGHYWA (SEQ ID NO: 872), HVR-H2, which comprises the sequence SISGYGSTTYYADSVKG (SEQ ID NO: 873), and HVR-H3, which comprises the sequence GGSDAVLGDWFAY (SEQ ID NO: 874). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQGIGSFLA (SEQ ID NO: 875), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 876), and HVR containing the sequence QQGYYLWT (SEQ ID NO: 877) Includes a light chain variable (VH) domain containing -L3.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1 comprising the sequence GFSLSTGVGVGVG (SEQ ID NO: 878), HVR-H2 comprising the sequence LIDWADDKYYSPSLKS (SEQ ID NO: 879), and HVR-H3 comprising the sequence GGSDTGIGDWFAY (SEQ ID NO: 880). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQSIGSYLA (SEQ ID NO: 881), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 882), and HVR containing the sequence QQGYYLWT (SEQ ID NO: 883). Includes a light chain variable (VH) domain containing -L3.

In some embodiments of any of the above embodiments, the antibodies of the present disclosure are multimeric antibodies (eg, bispecific antibodies). In some embodiments of any of the above embodiments, the antibody of the present disclosure is an IgM antibody and comprises, for example, an IgM Fc region (eg, a human IgM Fc region).

In another aspect, provided herein is a polynucleotide encoding either an antibody or antigen-binding fragment described herein. In some embodiments, provided herein is a polynucleotide comprising a sequence selected from SEQ ID NOs: 133-252.

In another aspect, provided herein is a vector containing any of the polynucleotides described above. In some embodiments, the vector is an expression vector.

In another aspect, provided herein are host cells containing either of the polynucleotides or vectors described herein (eg, bacterial cells, yeast cells, insect cells, mammalian cells (CHO cells or 293T). (Cells, etc.) etc.). In some embodiments, what is provided herein is a method of making an antibody or antigen-binding fragment, which comprises culturing a host cell under conditions suitable for producing the antibody or antigen-binding fragment. Is. In some embodiments, the method further comprises recovering an antibody or antigen binding fragment produced by a host cell.

In another aspect, provided herein is a pharmaceutical composition comprising any of the antibodies or antigen binding fragments (or derivatives thereof) described herein and a pharmaceutically acceptable carrier. ..

In another aspect, provided herein is a method of treating abnormal cell proliferation (eg, cancer) in a subject in need thereof, as described herein in therapeutically effective amounts. A method comprising administering to a subject any of an antibody, an antigen-binding fragment, and / or a pharmaceutical composition. In some embodiments, what is provided herein is a method of reducing tumor cell metastasis in a subject in a therapeutically effective amount of an antibody, antigen binding fragment, and / or medicament described herein. A method comprising administering any of the compositions to said subject. In some embodiments, the method is an additional treatment with a therapeutically effective amount of at least one (eg, at least one, at least two, at least three, at least four, at least five, at least ten, etc.). It further includes administering the drug to the subject. In some embodiments, at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapies, radiation therapy, and chemotherapy. In some embodiments, at least one additional therapeutic agent is pomalist, levrimid, renalide, pomalide, salidamide, DNA alkylated platinum-containing derivative, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD. -1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB -2 antibody, anti-EGFR antibody, setuximab, rituximab, trastuzumab, penbrolizumab, radiation therapy, single irradiation, divided irradiation, local irradiation, whole organ irradiation, IL-12, IFNα, GM-CSF, chimeric antigen acceptance It is selected from the group consisting of the body, adopted T cells, anti-cancer vaccines, and tumor-dissolving viruses. Also provided herein are the agents described herein for the treatment of abnormal cell growth (eg, cancer) and / or reduction of tumor cell metastasis in subjects in need thereof. Use of any of the compositions, antibodies, and / or antigen-binding fragments (or derivatives thereof). Also provided herein are books for the manufacture of pharmaceuticals for the treatment of abnormal cell growth (eg, cancer) and / or reduction of tumor cell metastasis in subjects in need thereof. Use of any of the antibodies or antigen-binding fragments (or derivatives thereof) described herein.

It should be appreciated that one, some, or all of the properties of the various embodiments described above and herein can be combined to form other embodiments of the present disclosure. These and other aspects of the disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description.

FIG. 1A shows a hypervariable region (HVR) definition compared to Kabat's CDR definition of an exemplary heavy chain variable region (VH) (SEQ ID NO: 13) and an exemplary light chain variable region (VL) (SEQ ID NO: 14). Is shown. FIG. 1B shows the selection of Fab hits that are cross-reactive with mouse CD137. An Elisa binding assay of exemplary antibodies to CD137 in humans, monkeys, and mice is shown. As shown at the top of the panels, each panel relates to a different antibody. FACS-based binding assay of exemplary antibodies to CD137 in humans, monkeys, mice, and rats is shown. As shown at the top of the panels, each panel relates to a different antigen. A comparison of species cross-reactivity between an exemplary antibody and a reference antibody is shown. Shown are exemplary antibodies that bind to activated human and monkey T cells but not to naive human T cells. The binding of AG10131 to activated human, monkey, mouse, and rat T cells is shown. It shows that the exemplary antibody has binding specificity for CD137 but not for other TNFR family members. 6A and 6B show that the exemplary antibody blocks the binding of CD137 and its cognate ligand CD137L by both ELISA (FIG. 6A) and flow cytometric assay (FIG. 6B). The results of epitope mapping by flow cytometry are shown. Multiple sequence alignments of parts of CD137 in humans (SEQ ID NO: 1), cynomolgus monkeys (SEQ ID NO: 860), and mice (SEQ ID NO: 861), along with CD137 sequences / regions of interest (annotated) identified from epitope mapping experiments. Shown. The agonistic activity of an exemplary antibody in the NFκB reporter assay is shown. Shows the agonistic activity of exemplary antibodies in CD8 + T cell proliferation (upper panel) and INF-γ secretion (lower panel). It shows the antitumor effect of an exemplary antibody in an H22 mouse liver cancer model and the infiltration of CD4 ⁺ and CD8 ⁺ T cells into the tumor. The antitumor effect of an exemplary antibody in a CT26 mouse colon cancer model is shown. The antitumor effect of an exemplary antibody in an EMT6 mouse breast cancer model is shown. It is shown that CT26 mice treated with the exemplary antibody remained tumor-free after re-challenge with the same tumor cells. It shows the killing of tumor cells by the splenocytes of re-challenge mice that rejected the tumor. It shows that AG10131 does not show ADCC effect. It shows that the exemplary antibody has a high concentration and low aggregation. Shows exemplary antibody stability under accelerated stress conditions. Shows thermal stability. AG10131 shows that normal mice are non-hematologically toxic up to 100 mg / kg (BIW) x 2 every other week. AG10131 shows that normal mice are free of histological liver abnormalities up to 100 mg / kg (BIW) x 2 every other week. AG10131 shows that cynomolgus monkeys are non-hematological at 10 mg / kg / week x 4. AG10131 shows that monkeys are not hepatotoxic at 10 mg / kg / week x 4. The pharmacokinetic profile of AG10131 in monkeys is shown. The pharmacokinetic profile of AG10131 in rats is shown. The pharmacokinetic profiles of various antibodies in mice are shown. The crystal structure of the human CD137-CD137L complex showing the CD137 cysteine rich domain (CRD) is shown. The epitope mapping of the shown anti-CD137 antibody binding to the shown human CD137 CRD by flow cytometry is shown. It is shown that the anti-CD137 antibody AG10131 blocks CD137 signaling stimulated by CD137L. The results showed that 293T cells stably expressing the NFκB luciferase reporter were transfected with a DNA construct expressing human CD137 and co-cultured with human B-cell lymphoma cells Daudi (upper) or Razi (lower) at the indicated ratios. The cellular NFκB luciferase reporter was then incubated overnight with a serial dilution of isotype control (left column) or ligand blocking anti-CD137 antibody (right column) (as shown) and then measured for luciferase activity. It is from the assay. It shows activation of human CD137-mediated NFκB signaling by anti-CD137 antibodies AG10131, AC1121, or AC1097 in the presence or absence of cross-linked antibodies. The EC50 (nM) of each antibody against NFκB signaling activation (with or without cross-linking) is shown. It shows that AG10131 and its human IgG4 isotype control antibody lack the ability to bind the human complement C1q component in the concentration range tested, whereas the human IgG1 isotype control antibody can bind to C1q. It shows the enhancement of tumor-invasive T lymphocytes by treatment with AG10131 in various tumor models. Upper left: Representative IHC-stained images of mouse CD4 + (upper panel) and CD8 + (lower panel) T cells in treated H22 tumor. Upper right: Representative IHC-stained images of mouse CD4 + (upper panel) and CD8 + (lower panel) T cells in treated EMT6 tumors. Lower center: Representative IHC-stained images of mouse CD4 + (upper panel) and CD8 + (lower panel) T cells in treated CT26 tumors. CD4 + and CD8 + T cells were stained black against the background of counterstaining of nuclei with hematoxylin. CD4 + and CD8 + T cells are indicated by black arrows. The number of tumor infiltrating T lymphocytes from the experiment shown in FIG. 31 is quantified. The percentages of CD4 ⁺ (upper) and CD8 ⁺ T cells (lower) in the tumors of H22, EMT6, and CT26 tumor samples were compared between the vehicle and the AG10131 treated group. **, p <0.01; ***, p <0.001. It shows the antitumor effect of AG10131 and anti-PD-1 antibody alone and in combination in an established mouse CT26 colon cancer syngeneic model. Top panel: Plot of mean tumor growth in each treatment group. Bottom panel: Spider plot of individual tumor growth in each group.

A. Definitions Unless otherwise defined herein, the terminology and technical terms used in connection with this disclosure shall have meanings commonly understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques used in connection with antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are: It is well known and commonly used in the art.

As used herein, the following terms have their associated meanings in this section.

The articles "a" and "an" refer to more than one or more (ie, at least one) grammatical object of the article. As an example, "an element" means one element or more than one element.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code and later modified amino acids, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" is a compound that has the same basic chemical structure as a naturally occurring amino acid, but with a C-terminal carboxy group, N-terminal amino group, or side chain functional group chemically modified to another functional group. Point to. The term "amino acid mimic" refers to a compound that has a structure different from the general chemical structure of an amino acid but that functions similarly to a naturally occurring amino acid.

The term "antibody" is used in the broadest sense herein and is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, a multispecific antibody (eg, two) as long as it exhibits the desired biological activity. (Heavy specific antibodies), and antibody fragments (eg, single chain variable fragments or scFv) are specifically included.

The term "antibody" is a term recognized in the art and has a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. It can refer to an antigen-binding protein (ie, immunoglobulin). Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. Each heavy chain has a variable region (abbreviated herein as _VH ) followed by a constant region at the N-terminus. The heavy chain constant region is comprised of three _domains, C _{H1, C H2,} and _{C H3.} Each light chain has a variable region (abbreviated as V _I herein) and the constant region of the other end subsequent to the N-terminus. The light chain constant region is comprised of one domain, C _L. V _L is parallel with the _{V H, C L} is parallel with the first constant domain of the heavy chain (CH1). The pairing of V _H and _VL both forms a single antigen binding site. IgM antibodies consist of 5 basic heterotetrameric units with an additional polypeptide called the J chain and thus contain 10 antigen binding sites, while secreted IgA antibodies polymerize. Together with the J chain, a multivalent aggregate consisting of 2 to 5 basic 4-chain units can be formed.

The V _H and _VL regions can be further subdivided into super variable regions called hypervariable regions (HVRs) based on structural analysis and sequence analysis. The HVR is interspersed with more conserved areas called framework areas (FW). For comparison, Kabat CDR definition by Yvonne Chen et al. (Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of AntiGen 881) are listed below (see also FIG. 1a). Each V _H and _VL consists of 3 HVRs and 4 FWs arranged in the following order from the amino terminus to the carboxy terminus: FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. Throughout the disclosure, the three heavy chain HVRs are referred to as HVR_H1, HVR_H2, and HVR_H3. Similarly, the three HVRs of the light chain are called HVR_L1, HVR_L2, and HVR_L3.

The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q). Within the light and heavy chains, the variable and constant regions are linked by the "J" region of about 12 or more amino acids, and the heavy chain also contains the "D" region of about 10 or more amino acids. In general, Fundamental Immunology Ch. ^{7 (Paul, W., ed.} , 2 nd ed. Raven Press, N.Y. (1989)) , incorporated herein by reference.

L chains from any vertebrate can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domain. Antibodies can be assigned to different classes or isotypes, depending on the amino acid sequence of their heavy chain constant domain (CH). There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, heavy chains called α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. Has. The IgG classes of antibodies can be further classified into four subclasses IgG1, IgG2, IgG3, and IgG4, respectively, by gamma heavy chains Y1 to Y4.

The term "antibody derivative" or "derivative" of an antibody is a molecule that can bind to the same antigen to which the antibody binds (eg, CD137) and contains the amino acid sequence of the antibody linked to additional molecular entities. Point to. The amino acid sequence of the antibody contained in the antibody derivative is the full-length heavy chain, the full-length light chain, any part or multiple parts of the full-length heavy chain, any part or multiple parts of the full-length light chain, or the antibody. It can be any other fragment (s), or a complete antibody. The additional molecular entity can be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (enzymes, antibodies, etc.), and compounds. Additional molecular entities may have any utility, such as use as a detector, label, marker, pharmaceutical, or therapeutic agent. The amino acid sequence of an antibody may be bound or linked to additional molecular entities by chemical coupling, genetic fusion, non-covalent bonding, or other methods. The term "antibody derivative" also includes molecules derived from modification of the amino acid sequence of the CD137 antibody, such as chimeric antibodies, humanized antibodies, and substitutions, additions, and insertions of conserved amino acids.

The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more parts of an antibody that retains the ability to bind to the antigen to which the antibody binds (eg, CD137). Examples of "antigen-binding fragment" of an _antibody, Fab fragment, a monovalent fragment consisting of _{(i) V L, V H} , C L, and _{C H1} domains; linked by a disulfide bridge at (ii) the hinge region F (ab') ₂ fragments, which are divalent fragments containing two Fab fragments; Fd fragments consisting of (iii) V _H and _CH1 domains; (iv) consisting of single arm _VL and _VE domains of antibodies. Fv fragments, (v) dAb fragments consisting of _VH domains (Ward et al., Nature 341: 544-546 (1989)); (vi) isolated complementarity determining regions (CDRs) are included.

The term "binding molecule" includes (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) derivatives of antibodies, each as defined herein.

The terms "binding to CD137", "binding to CD137", "binding to CD137", or "binding to CD137" are used in Example 4 of the binding molecule as defined herein. in in vitro assays, such as Biacore assays as described in, it refers to binding to human CD137 at 100nM or less of affinities _{(K D).}

The terms "CD137" and "CD137 receptor" are used interchangeably herein and include the human CD137 receptor and its variants, isoforms, and species homologues. Thus, the binding molecules defined and disclosed herein can also bind to CD137 from species other than humans. In other cases, the binding molecule may be completely specific for human CD137 and may not exhibit species or other types of cross-reactivity.

The term "CD137 antibody" refers to an antibody capable of binding to the human CD137 receptor, as defined herein.

The term "chimeric antibody" refers to an antibody comprising an amino acid sequence derived from a different animal species, such as one having a variable region derived from a human antibody and a mouse immunoglobulin constant region.

The term "competing for binding" refers to the interaction of two antibodies in binding to their binding target. If the binding of the primary antibody to its cognate epitope is detectably reduced in the presence of the secondary antibody compared to the binding of the primary antibody in the absence of the secondary antibody, then the primary antibody is with the secondary antibody for binding. Competing. Alternatively, if the binding of the secondary antibody to its epitope is also detectably reduced in the presence of the primary antibody, this is not always the case, but it is possible. That is, the primary antibody can inhibit the binding of the secondary antibody to its epitope without the secondary antibody inhibiting the binding of the primary antibody to each epitope. However, if each antibody detectably inhibits the binding of another antibody to its cognate epitope, then to the same extent, greater than or equal to, the antibody will exhibit for the binding of each epitope (s). They are said to be "cross-competitive" with each other.

The term "epitope" refers to the portion of an antigen to which an antibody (or its antigen-binding fragment) binds. Epitopes can be formed from both continuous or discontinuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from continuous amino acids are typically retained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes can contain varying numbers of amino acids with unique spatial conformations. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium hydrogen exchange combined with mass spectrometry, or site-specific mutagenesis, or antigens and their bound antibodies and All methods used in combination with computational modeling of complex structures with that variant are included. For example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). Once the desired epitope of the antigen has been determined, for example, the techniques described herein can be used to generate antibodies against that epitope. Antibody production and characterization can provide information about the desired epitope. From this information, antibodies can be screened competitively for binding to the same epitope. The approach to achieving this is to perform cross-competitive studies to find antibodies that bind competitively to each other, ie, antibodies that compete for binding to the antigen. A high-throughput process for "binning" antibodies based on mutual competition is described in PCT Publication No. WO 03/48731.

The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as they are passed from parent to offspring via germ cells. The germline sequence is distinguished from the nucleotide sequence encoding the antibody of mature B cells that has been altered by recombination and high frequency mutation events during B cell maturation.

The term "glycosylation site" refers to an amino acid residue that is recognized by eukaryotic cells as an additional position for a sugar residue. Amino acids added by carbohydrates such as oligosaccharides are typically asparagine (N bond), serine (O bond), and threonine (O bond) residues. Specific addition sites are typically indicated by an amino acid sequence referred to herein as the "glycosylation site sequence." The glycosylation site sequence of N-linked glycosylation is as follows: -Asn-X-Ser- or -Asn-X-Thr-, where X is any of the standard amino acids other than proline. Good. The terms "N bond" and "O bond" refer to a chemical group that functions as an addition site between a sugar molecule and an amino acid residue. The N-linked sugar is added via an amino group, and the O-linked sugar is added via a hydroxyl group. The term "glycan occupancy" refers to the presence of a sugar moiety linked to a glycosylation site (ie, glycan site is occupied). If there are at least two potential glycosylation sites on the polypeptide, either none (0-glycan site occupancy), one (1-glycan site occupancy), or both (2-glycan site occupancy) Can be occupied by the sugar moiety.

The term "host cell" refers to a cell line that can be genetically engineered to produce the protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, mammals derived from rodents (rat, mouse, guinea pig, or hamster) such as CHO, BHK, NSO, SP2 / 0, YB2 / 0, or human tissue or hybridoma cells. Includes cultured animal cells, yeast cells, insect cells, and cultured cells such as transgenic animals or cells contained within cultured tissues. The term includes not only specific cells of interest, but also the progeny of such cells. Such progeny may not be identical to the parent cell, as certain modifications may occur in subsequent generations due to mutations and environmental influences, but are still within the term "host cell". include.

The term "human antibody" refers to an antibody in which the entire amino acid sequence of the light and heavy chains is derived from a human immunoglobulin gene. When produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, the human antibody may comprise a mouse sugar chain. Human antibodies can be prepared by a variety of methods known in the art.

The term "humanized antibody" refers to a chimeric antibody that contains an amino acid residue derived from a human antibody sequence. Humanized antibodies can include some or all of CDRs or HVRs from non-human animal or synthetic antibodies, and the framework and constant regions of the antibody include amino acid residues from the human antibody sequence.

The term "exemplary antibody" refers to any one of the antibodies described in the present disclosure and designated as listed in Tables 1a and 1b. These antibodies may be of any class (eg, IgA, IgD, IgE, IgG, and IgM). Thus, each antibody identified above includes all five classes of antibodies that have the same amino acid sequence for the _VL and _VH regions. In addition, IgG class antibodies can be of any subclass (eg, IgG1, IgG2, IgG3, and IgG4). Thus, each antibody of the IgG subclass identified above includes antibodies of all four subclasses having the same amino acid sequence for the _VL and _VH regions. The amino acid sequences of the heavy chain constant regions of human antibodies of the 5 classes and 4 IgG subclasses are known in the art. The amino acid sequences of the full-length heavy and light chains of each of the IgG4 subclasses of the exemplary antibodies shown in Table 1b are provided herein.

The term "isolated antibody" or "isolated binding molecule" refers to an antibody or binding molecule as defined herein and is (1) naturally associated with it in its native state. Not associated with an ingredient; (2) free of other proteins from the same species; (3) expressed by cells of different species; or (4) non-naturally occurring. Examples of isolated antibodies include CD137 antibodies that have been affinity purified using CD137, CD137 antibodies produced by hybridomas or other cell lines in vitro, and CD137 antibodies derived from transgenic animals. ..

The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, isolated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules isolated from the chromosomes to which the genomic DNA is naturally associated. Preferably, the "isolated" nucleic acid does not contain sequences that are naturally adjacent to the nucleic acid (ie, sequences located at the 5'and 3'ends of the nucleic acid of interest).

The term "k _a", the specific antibody - refers to an association rate constant of the antigen interaction, the term "k _d", the specific antibody - refers to the dissociation rate constant for antigen interaction.

The term "K _D", a particular antibody - refers to the equilibrium dissociation constant of antigen interaction. This is obtained from the ratio (i.e. _k d / _{k a)} for _{k a} of _{k d,} expressed as a molar concentration (M). _The K _D is used as a measure of the affinity of the antibody that binds to the binding partner. As a K _D The smaller, the antibody or binds more strongly, or antibody and the affinity between the antigen increases. For example, an antibody with a nanomolarity (nM) dissociation constant binds more strongly to a particular antigen than an antibody with a micromolarity (μM) dissociation constant. K _D of an antibody can be determined using methods well established in the art. One method for determining the K _D of an antibody is typically using a biosensor system such as a Biacore (R) system, by using a surface plasmon resonance. Assay procedures using the BIACORE ™ system (BIAcore assay) are described in the Examples section of this disclosure.

The term "mammal" refers to any species of mammal in the class. Examples of mammals include: laboratory animals such as rats, mice, monkeys, and guinea pigs; livestock such as cats, dogs, rabbits, cows, sheep, goats, horses, and pigs; as well as lions, tigers, elephants. Wild animals in captivity such as.

The term "preventing" or "preventing" with respect to a particular disease state in a mammal refers to preventing or delaying the onset of the disease, or preventing the onset of its clinical or asymptomatic symptoms.

As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Amino acid sequence identity of a polypeptide can be conventionally determined using a known computer program such as Bestfit, FASTA, or BLAST (eg, Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods). Mol. Biol. 132: 185-219 (2000); Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nuclear Acids Res. 25: 3389-3402 (1997). Please refer to). When using Bestfit or other sequence alignment programs to determine if a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are calculated with the same percentage over the entire length of the reference amino acid sequence. A homology gap of up to 5% of the total number of amino acid residues in the reference sequence is set to be acceptable. This aforementioned method of determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

As defined herein, with respect to the interaction of a binding molecule (eg, an antibody) with its binding partner (eg, an antigen), it is referred to as "specifically binding" or "specifically binding to". The term refers to the ability of a binding molecule to distinguish an antigen of interest from an animal species from an antigen ortholog from a different animal species under certain conditions. A CD137-binding molecule is said to specifically bind to human CD137 if it binds to human CD137 with less than 50% of the EC50 that binds to rat or mouse CD137, as determined by the in vitro assay. The binding specificity of an antibody can be determined using methods known in the art. Examples of such methods include FACS, Western blots, ELISA, RIA, ECL, IRMA tests, and peptide scans using PHA-stimulated primary cells.

As defined herein, with respect to the interaction of a binding molecule (eg, an antibody) with its binding partner (eg, an antigen), it is referred to as "selectively binding" or "selectively binding to". The term refers to the ability of a binding molecule to discriminate between a target antigen from an animal species (such as human CD137) and a different antigen from the same animal species (such as human CD40) under predetermined conditions. CD137-binding molecules are said to selectively bind to human CD137 if they bind to human CD137 with less than 10% of EC50 that binds to human CD40 or human CD134, as determined by in vitro assay.

The terms "treat," "treat," or "treat" with respect to a particular disease state of a mammal refer to causing a desirable or beneficial effect on the mammal with the disease state. Desirable or beneficial effects include a reduction in the frequency or severity of one or more symptoms of the disease (ie, tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells, etc.). , Or may include stopping or inhibiting further progression of the disease, condition, or disorder. In the context of cancer treatment in mammals, desirable or beneficial effects include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of recurrence of cancer, reduction of pain associated with cancer. , Or an improvement in mammalian survival. The effect can be either subjective or objective. For example, if the mammal is a human, the human may point to an improvement in vitality or vitality, or a reduction in pain, as a subjective symptom of improved treatment or response. Alternatively, clinicians may point to a decrease in tumor size or tumor volume based on physical examination, laboratory values, tumor markers, or x-ray findings. Some laboratory signs that clinicians may observe about their response to treatment include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. .. In addition, clinicians may observe a decrease in detectable tumor markers. Alternatively, other tests such as sonograms, nuclear magnetic resonance tests, and positron emission tests can be used to evaluate objective improvements.

The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by a recombination technique such as ligation or recombination. This allows foreign nucleic acid molecules to be propagated, selected, further manipulated or expressed in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. One type of vector integrates into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome (eg, non-episome mammalian vector). Another type of vector is capable of autonomous replication in the host cell into which it has been introduced (eg, a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). Another particular type of vector capable of inducing the expression of an expressible foreign nucleic acid to which they are operably linked is commonly referred to as an "expression vector". Expression vectors generally have control sequences that drive the expression of expressible foreign nucleic acids. Simpler vectors known as "transcriptional vectors" can only be transcribed and cannot be translated: they can replicate in target cells but are not expressed. The term "vector" includes all types of vectors, regardless of their function. Vectors capable of inducing the expression of expressible nucleic acids to which they are operably linked are commonly referred to as "expression vectors".

The methods and techniques of the present disclosure are generally practiced in accordance with methods well known in the art and are described in various general and more specific references cited and discussed throughout this specification unless otherwise stated. .. Such references are described, for example, in Sambrook and Russel, Molecular Cloning, A Laboratory Application, Cold Spring Harbor Press, Cold Spring Harbor, N. et al. Y. (2001), Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Collor Bors, Coll. Y. (1990) is included. Enzymatic reaction and purification techniques are performed according to the manufacturer's specifications as commonly achieved in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry and pharmaceutical chemistry, as described herein, as well as laboratory procedures and techniques are well known and commonly used in the art. It is a thing. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and treatment of patients.

As used herein, the 20 conventional amino acids and their abbreviations follow standard usage. See Immunology-A Synthesis (2nd Edition, E.S. Goruband DR Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

B. Binding Molecules Binding to Human CD137 The present disclosure provides isolated binding molecules that bind to human CD137, including CD137 antibodies, antigen binding fragments of CD137 antibodies, and derivatives of CD137 antibodies. In some embodiments, the binding molecule is described in relation to the antibodies described in relation to epitope binding, and HVR, variable regions (VL, VH), and IgG (eg, IgG4) light and heavy chains. Any of the antibodies that have been produced. In some embodiments, the present disclosure binds to human CD137 and at least one of the following functional properties (eg, at least one, at least two, at least three, at least four, at least five). For binding molecules having at least 6, at least 7, 8, or all 9): (a) binds to human CD137 with a KD of 500 nM or less; (b) has agonistic activity against human CD137; (c) Does not bind to human OX40, CD40, GITR, and / or CD27 receptors at concentrations up to 1000 nM; (d) cross-reactive with monkey, mouse, rat, or canine CD137; (e) induces ADCC effect No; (f) can inhibit the growth of tumor cells; (g) has a therapeutic effect on cancer; (h) blocks the binding between CD137 and CD137L; and (i) in cells expressing CD137, CD137L Blocks CD137 signaling stimulated by (eg, NF-κB-dependent transcription stimulated by CD137L). In some embodiments, the antibodies disclosed herein can also block the binding between CD137 and its ligand CD137L, eg, completely. Also provided herein are one or more anti-CD137 antibodies or antigen-binding fragments that cross-compete with one or more of the antibodies or antigen-binding fragments described herein for binding to human CD137. ..

In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment binds to one or more amino acid residues within amino acid residues 34-93 of SEQ ID NO: 1. In some embodiments, the antibody or antigen binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO: 1. .. In some embodiments, the antibody or antigen binding fragment of SEQ ID NO: 1 is one or more of amino acid residues 34-36, one or more of 53-55, and one of 92-93. Combine one or more. In some embodiments, the antibody or antigen binding fragment is among the amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. Do not combine into one or more. In some embodiments, the antibody or antigen binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. Methods of measuring the ability of an antibody or antigen-binding fragment to bind to a target antigen include any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, and the like. It may be carried out using. In some embodiments, the ability of an antibody or antigen-binding fragment to bind to a target antigen is measured by surface plasmon resonance (see, eg, Example 1 below).

In some embodiments, the antibody or antigen binding fragment is a KD of about 500 nM or less (eg, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, About 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc. ), It binds to human CD137. In some embodiments, the antibody or antigen binding fragment binds to human CD137 at a KD of about 100 nM or less. In some embodiments, the antibody or antigen binding fragment binds to human CD137 at a KD of about 50 nM or less. Methods for measuring the KD of an antibody or antigen-binding fragment are performed using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, and the like. You may. In some embodiments, KD is measured by surface plasmon resonance (see, eg, Example 1 below).

The anti-CD137 antibody needs to be crosslinked to become an agonist. For example, cross-linking is achieved in vivo via the Fc gamma receptor, but typically, polyclonal anti-Fc antibodies are used in cell-based experiments in vitro. In some embodiments, the antibodies or antigen-binding fragments described herein have agonistic activity against human CD137. In some embodiments, the antibody or antigen-binding fragment is one or more (eg, one or more) of human CD137 when a cell expressing human CD137 (eg, human cell) comes into contact with the antibody or antigen-binding fragment. (2, 2 or more, 3 or more, etc.) of activity is induced. Various CD137 activities are known in the art and, without limitation, induction of NF-κB-dependent transcription, induction of T cell proliferation, prolongation of T cell survival, co-stimulation of activated T cells, cytokine secretion ( It may include induction of IL-2 etc.) and induction of monocyte activation. In some embodiments, one or more CD137 activities are not CD137 binding to its ligand. Methods for measuring CD137 activity (eg, induction of NF-κB-dependent transcription and / or T cell proliferation) are known in the art and are described, for example, by the methods described in Examples 8 and 9 below. Including. In some embodiments, the antibody or antigen-binding fragment increases NF-κB-dependent transcription in cells expressing human CD137 (eg, human cells). In some embodiments, NF-κB-dependent transcription is performed in cells expressing CD137 that are in contact with an antibody or antigen-binding fragment (eg, human cells) and corresponding cells that are not in contact with the antibody or antigen-binding fragment (eg, human cells). For example, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, as compared to (corresponding cells that are not in contact with the antibody or are in contact with the isotype control antibody). It increases by about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 99% or more. In some embodiments, NF-κB-dependent transcription is performed in cells expressing CD137 that are in contact with an antibody or antigen-binding fragment (eg, human cells) and corresponding cells that are not in contact with the antibody or antigen-binding fragment (eg, human cells). For example, about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, compared to (corresponding cells that are not in contact with the antibody or in contact with the isotype control antibody). Increases 10-fold, 100-fold, 1000-fold, or more.

In some embodiments, the antibody or antigen binding fragment is cross-reactive with monkeys (eg, cynomolgus monkeys), mice, rats, and / or dog CD137. In some embodiments, the antibody or antigen binding fragment is cross-reactive with monkey CD137. In some embodiments, the antibody or antigen binding fragment is cross-reactive with mouse CD137. In some embodiments, the antibody or antigen binding fragment is cross-reactive with rat CD137. In some embodiments, the antibody or antigen binding fragment is cross-reactive with canine CD137. In some embodiments, the antibody or antigen binding fragment is a monkey and mouse CD137; a monkey and a rat CD137; a monkey and a dog CD137; a mouse and a dog CD137; a mouse and a dog CD137; a rat and a dog CD137; It is cross-reactive with monkey, mouse, and rat CD137; monkey, mouse, and dog CD137; monkey, rat, and dog CD137; mouse, rat, and dog CD137; or monkey, mouse, rat, and dog CD137. In some embodiments, the antibody or antigen binding fragment is cross-reactive at about 100 nM (eg, about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM). Methods for measuring antibody cross-reactivity, including, but not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc., are known in the art. In some embodiments, cross-reactivity is measured by ELISA (see, eg, Example 2 below).

In some embodiments, the antibody does not elicit an ADCC effect. Non-limitingly, methods for measuring ADCC effects (eg, in vivo methods), including the methods described in Example 11 below, are known in the art. In some embodiments, the antibody does not induce more than about 10% ADCC effect compared to the control (more than about 10%, more than about 5%, more than about 1%, about 0.1%). Exceeds, exceeds about 0.01%, does not induce ADCC).

In some embodiments, the antibody or antigen-binding fragment can inhibit the growth / proliferation of tumor cells. In some embodiments, tumor cell growth / proliferation, when in contact with an antibody or antigen-binding fragment, is compared to the corresponding tumor cell that is not in contact with the antibody or antigen-binding fragment. At least about 5% (eg, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80 %, At least about 90%, or at least about 99%). In some embodiments, the antibody or antigen-binding fragment can reduce the tumor volume of a subject when the antibody or antigen-binding fragment is administered to the subject. In some embodiments, the antibody or antigen binding fragment is at least about 5% (eg, at least about 5%, at least) compared to the initial tumor volume in the subject (eg, before administration of the antibody or antigen binding fragment). About 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) , Reduce. Methods of monitoring tumor cell growth / growth, tumor volume, and / or tumor inhibition are known in the art, including, for example, by the method described in Example 10 below. is there.

In some embodiments, the antibody or antigen binding fragment has a therapeutic effect on cancer. In some embodiments, the antibody or antigen binding fragment alleviates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer is in partial or complete remission upon administration of an antibody or antigen binding fragment.

In another aspect, the disclosure provides isolated antibodies that compete or cross-competition with any of the exemplary antibodies of the disclosure, such as AG10048, AG10059, and / or AG10131 for binding to human CD137. In certain embodiments, the disclosure provides isolated antibodies that compete or cross-competition with any of the exemplary antibodies of the disclosure for binding to the same epitope on human CD137. The ability of an antibody to compete or cross-competition for binding to another antibody can be determined using standard binding assays known in the art such as BIAcore analysis, ELISA assay, or flow cytometry. For example, the exemplary antibodies of the present disclosure can allow binding to human CD137 under saturated conditions, and then the ability of the test antibody to bind to CD137 can be measured. If the test antibody can bind to CD137 at the same time as the exemplary antibody, the test antibody binds to an epitope different from the exemplary antibody. However, if the test antibody cannot bind to CD137 at the same time, the test antibody binds to the same epitope, overlapping epitope, or epitope very close to the epitope to which the exemplary antibody binds. This experiment can be performed using a variety of methods such as ELISA, RIA, FACS, or surface plasmon resonance.

In some embodiments, the antibody or antigen binding fragment blocks the binding between CD137 and its ligands (eg, human CD137 and human CD137L). In some embodiments, the antibody or antigen binding fragment blocks the binding between CD137 and its ligand in vitro. In some embodiments, the antibody or antigen binding fragment is about 500 nM or less (eg, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, for blocking the binding of its ligand to CD137. It has a half-inhibition concentration (IC50) of about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.). In some embodiments, the antibody or antigen binding fragment has a half inhibitory concentration (IC50) of about 100 nM or less for blocking the binding of its ligand to CD137. In some embodiments, the antibody or antigen binding fragment is its ligand for human CD137 when provided at a concentration of about 100 nM or higher (eg, about 100 nM or higher, about 500 nM or higher, about 1 μM or higher, about 10 μM or higher, etc.). Completely block the binding to. As used herein, the term "completely blocking" or "completely blocking" refers to at least about 80% of the binding of an antibody or antigen-binding fragment between a first protein and a second protein (as used herein). For example, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.), refers to the ability to reduce. Methods of measuring the ability of an antibody or antigen binding fragment to block the binding of a first protein (eg, CD137) and a second protein (eg, CD137L) include, but are not limited to, BIAcore analysis, ELISA assay, and flow cytometry. It is known in the art (see, eg, Example 6 below).

B-1. CD137 Antibody In some embodiments, the disclosure provides an isolated antibody that binds to human CD137 at an epitope within amino acid residues 34-108 or 34-93 of SEQ ID NO: 1. In some embodiments, the antibody, as measured by surface plasmon resonance, binds to human CD137 with the following _{K D} 50 nM. In certain embodiments, the antibody can be cross-reactive with at least one non-human species selected from the list consisting of cynomolgus monkeys, mice, rats, and dogs.

In one aspect, the present disclosure provides an isolated antibody comprising a heavy chain variable region and a light chain variable region, a) the heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3. HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (I): X1TFX2X3YX4IHWV (SEQ ID NO: 2), in which X1 is F or Y and X2 is S or T. , X3 is G, N, or S, and X4 is A, G, or W; formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 3), where X1 is S or T and X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO: 4), where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T; HVR-H2 comprises an amino acid sequence according to a formula selected from the group: formula ( IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO: 5), in the formula, X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T. Yes; formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO: 6), in the formula, X1 is D or E, X2 is N or S, and X3 is N or S; and formula (VI): VSX1ISGX2GX3X4TYYADSVGRF SEQ ID NO: 7), in the equation, X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S. Or T; and HVR-H3 comprises the amino acid sequence according to formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO: 8), in which X1 is E or G, X2 is E or S and X3 is D. Or T, X4 is A, T, or V, X5 is A, I, L, T, or V, and X6 is A, D, or G; and / or b) light chain. The variable region comprises HVR-L1, HVR-L2, and HVR-L3, where HVR-L1 comprises the amino acid sequence according to formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO: 9), where X1 is Q or R. , X2 is D, G, or S, X 3 is I or V, X4 is G, R, S, or T, X5 is P, R, S, or T, and X6 is A, D, F, S, V, or Y. , X7 is L or V, and X8 is A, G, or N; HVR-L2 comprises the amino acid sequence according to formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO: 10), where X1 is A or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V. And HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (X): YCQQX1YX2X3X4T (SEQ ID NO: 11), in which X1 is A, G, S or Y. X2 is Q, S, or Y, X3 is I, L, T, or Y, and X4 is I, S, V, or W; and formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO: 12). , In the formula, X1 is E or Q, X2 is P, S or Y, X3 is D, L, S, T or Y and X4 is D, E, H, S or T. X5 is D, L, T, or W, and X6 is L, P, R, or V.

In some embodiments, the antibody is HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 253 to 312, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 313 to 372, HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 373 to 432, HVR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 492, and selecting from the group consisting of SEQ ID NOs: 493 to 552. It may include HVR-L2 having an amino acid sequence to be prepared, and HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 553 to 612.

In certain embodiments, the antibody has a VL and an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-132, each of which is preferably encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 133-252. / Or may include VH.

In some embodiments, the antibody is HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 709 to 732, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 733 to 756, Select from the group consisting of HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 757 to 780, HVR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 781 to 804, and SEQ ID NOs: 805 to 828. It may include HVR-L2 having an amino acid sequence to be prepared, and HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 829 to 852.

In certain embodiments, the antibody is a light chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 613-660, each of which is preferably encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 661-708. And / or heavy chains (eg, those of IgG such as IgG4) may be included.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1 containing the sequence GFSLSTGVGVG (SEQ ID NO: 866), HVR-H2 containing the sequence LIDWDDDKYSPSLKS (SEQ ID NO: 867), and HVR-H3 containing the sequence GGSDTLVDWFAY (SEQ ID NO: 868). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQSVSPYLA (SEQ ID NO: 869), HVR-L2 containing the sequence DASSLES (SEQ ID NO: 870), and HVR containing the sequence QQGYSLWT (SEQ ID NO: 871). Includes a light chain variable (VH) domain containing −L3.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1, which comprises the sequence GYSITSGHYWA (SEQ ID NO: 872), HVR-H2, which comprises the sequence SISGYGSTTYYADSVKG (SEQ ID NO: 873), and HVR-H3, which comprises the sequence GGSDAVLGDWFAY (SEQ ID NO: 874). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQGIGSFLA (SEQ ID NO: 875), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 876), and HVR containing the sequence QQGYYLWT (SEQ ID NO: 877) Includes a light chain variable (VH) domain containing -L3.

In some embodiments, the HVR is by Kabat. In some embodiments, the antibody comprises HVR-H1 comprising the sequence GFSLSTGVGVGVG (SEQ ID NO: 878), HVR-H2 comprising the sequence LIDWADDKYYSPSLKS (SEQ ID NO: 879), and HVR-H3 comprising the sequence GGSDTGIGDWFAY (SEQ ID NO: 880). Heavy chain variable (VH) domain comprising; and / or HVR-L1 containing the sequence RASQSIGSYLA (SEQ ID NO: 881), HVR-L2 containing the sequence DASNLET (SEQ ID NO: 882), and HVR containing the sequence QQGYYLWT (SEQ ID NO: 883). Includes a light chain variable (VH) domain containing -L3.

The CD137 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA, or IgD. The CD137 antibody is preferably of an IgG class such as IgG1, IgG2, IgG3, or IgG4 subclass. The CD137 antibody can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody in a desired class or subclass is the step of isolating the nucleic acid encoding the heavy chain of the CD137 antibody and the nucleic acid encoding the light chain of the CD137 antibody, and encoding the _VH region. A step of isolating the sequence, a step of linking the _VH sequence to a sequence encoding a heavy chain constant region of a desired class or subclass, a step of expressing a light chain gene and a heavy chain construct in a cell, and a CD137 antibody. Includes steps to collect.

In addition, the antibodies provided by the present disclosure can be monoclonal or polyclonal, but are preferably monoclonal.

Examples of specific isolated antibodies provided by the present disclosure include those listed in Tables 1a and 1b. The nucleotide and amino acid sequences of the heavy chain variable regions of these antibodies, the full length heavy chains of the IgG2 and IgG4 subclasses, the light chain variable regions, and the full length light chains are also provided below.

The antibodies of the present disclosure are transformed by a viral or tumor gene in B lymphocytes, see conventional monoclonal antibody methodologies, eg, standard somatic cell hybridization techniques (eg, Kohler and Milstein, Nature 256: 495 (1975)). Or can be produced by techniques known in the art, including recombinant antibody techniques described in detail below herein.

Hybridoma production is a very well-established procedure. A common animal system for preparing hybridomas is the mouse system. Techniques for the isolation of immunospleen cells for immunization protocols and fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion procedures are also known. One well-known method that can be used to make the human CD137 antibody provided by the present disclosure comprises the use of the XenoMouse ™ animal system. XenoMouse ™ mice are genetically engineered mouse strains that contain large fragments of human immunoglobulin heavy and light chain loci and lack mouse antibody production. For example, Green et al. , Nature Genetics 7: 13-21 (1994) and WO 2003/040170. Animals are immunized with the CD137 antigen. The CD137 antigen is isolated and / or purified CD137, preferably CD137. It may be a fragment of CD137, such as the extracellular domain of CD137, in particular a CD137 extracellular domain fragment comprising amino acid residues 34-108 or 34-93 of SEQ ID NO: 1. Immunization of animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cows, and horses are well known in the art. See, for example, Harlow and Lane, supra, and US Pat. No. 5,994,619. The CD137 antigen may be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulatory complex). After immunizing an animal with the CD137 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and the lymph nodes and / or spleen B cells are immortalized. Methods of immortalizing cells include, but are not limited to, introducing oncogenes into them, infecting them with tumor viruses, culturing them under conditions that select immortalized cells, and carcinogenicizing them. Includes serving with sex or mutant compounds, fusing with immortalized cells such as myeloma cells, and inactivating tumor suppressor genes. See, for example, the Harlow and Line above. When fusion with myeloma cells is used, myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secretory cell lines). Immortalized cells are screened using CD137, a portion thereof, or cells expressing CD137. CD137 antibody-producing cells, such as hybridomas, are selected, cloned, and further screened for desirable properties, including strong proliferation, high antibody production, and desirable antibody properties, as further discussed below. Hybridomas can be grown in in vivo or in vitro cell cultures in syngeneic animals, animals lacking the immune system, such as nude mice. Methods of selecting, cloning, and propagating hybridomas are well known to those of skill in the art.

The antibodies of the present disclosure can also be prepared using the phage display method or the yeast display method. Such display methods for isolating human antibodies are described in Achim Knappik, et al. , "Fully Synthetic Human Combinatorial Antibodies (HuCAL) Based on Modern Consensus Frameworks and CDRs Randomized with Jits Trintle. Mol. Biol. (2000) 296, 57-86; and Michael J. et al. Felhaus, et al, "Flow-cytometry isolation of human antibodies from a non-immune Saccharomyces cerevisiae cerevisiae cerevisiae selevisiae surface sen lay ri

B-2. Antigen-Binding Fragment In some other aspects, the disclosure provides an antigen-binding fragment of any of the CD137 antibodies provided by the present disclosure.

The antigen binding fragment may contain any sequence of antibodies. In some embodiments, the antigen-binding fragment comprises the following amino acid sequences: (1) the light chain of the CD137 antibody; (2) the heavy chain of the CD137 antibody; (3) the variable region derived from the light chain of the CD137 antibody; 4) Variable region derived from the heavy chain of the CD137 antibody; (5) one or more HVRs (2, 3, 4, 5, or 6 HRVs) of the CD137 antibody; or (6) 3 derived from the light chain of the CD137 antibody. One HVR and three HVRs derived from heavy chains.

In some specific embodiments, the present disclosure provides antigen-binding fragments of antibodies selected from those listed in Tables 1a and 1b.

In another particular embodiment, the antigen binding fragment of CD137 _{antibody, (i) V L, V} H, C L, or Fab fragment, a monovalent fragment consisting of _{C H} 1 domains; with (ii) the hinge region a bivalent fragment comprising two Fab fragments linked by a disulfide bridge F (ab _{') 2} fragments; (iii) _{V H} and _{C H} consists of one domain Fd fragment; (iv) V of a single arm of an antibody Fv fragment consisting of _L and _VH domains, (v) dAb fragment consisting of _VH domain (Word et al., (1989) Nature 341: 544-546); (vi) Isolated complementarity determining regions (CDR) ); And (vii) are single chain antibodies (scFv), which are polypeptides comprising the _VL region of the antibody linked to the _VH region of the antibody. Bird et al. , (1988) Science 242: 423-426, and Huston et al. , (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883.

In some specific embodiments, the antigen binding fragment is a Fab fragment selected from those listed in Table 1a.

B-3. Antibody Derivatives In some further embodiments, the present disclosure provides derivatives of any of the CD137 antibodies provided by the present disclosure.

In one aspect, the antibody derivative is derived from modifying the amino acid sequence of the exemplary antibody of the disclosure (“parent antibody”), preserving the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain, such as the framework region, HVR region, or constant region, may be modified. Types of modifications include substitutions, insertions, deletions, or combinations thereof of one or more amino acids of the parent antibody.

In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequences set forth in SEQ ID NOs: 13-132. Includes a _VL or _VH region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 253 to 312. Includes an HVR_H1 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 313-372. Includes an HVR_H2 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 373-432. Includes an HVR_H3 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative comprises the HVR_L1 amino acid sequences of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 433-492. In some embodiments, the antibody derivative comprises the HVR_L2 amino acid sequences of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 493 to 552.

In some embodiments, the antibody derivative comprises the HVR_L3 amino acid sequence of all Fab hits shown in Table 1a, which can be found in SEQ ID NOs: 553-612. In some specific embodiments, the derivative is 1, 2, 3, 4, 5, 6, 7, with respect to the amino acid sequence set forth in any of SEQ ID NOs: 13-132, and 253-612. 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Includes 11, 12, 13, 14, or 15 additions and / or deletions.

In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 613-660. Includes light or heavy chains that are at least 97%, at least 98%, or at least 99% identical.

In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 709-732. Includes an HVR_H1 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 733-756. Includes an HVR_H2 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 758-780. Includes an HVR_H3 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 781-804. Includes an HVR_L1 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NO: 805-828. Includes an HVR_L2 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the antibody derivative is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the amino acid sequence set forth in SEQ ID NOs: 829-852. Includes an HVR_L3 amino acid sequence region that is at least 97%, at least 98%, or at least 99% identical. In some specific embodiments, the derivative is 1, 2, 3, 4, 5, 6, 7, with respect to the amino acid sequence set forth in any of SEQ ID NOs: 613-660, and 709-852. 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Includes 11, 12, 13, 14, or 15 additions and / or deletions.

Amino acid substitutions include both conservative and non-conservative substitutions. The term "conservative amino acid substitution" refers to the similarity of two amino acids in certain physicochemical properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. Means to replace one amino acid with another amino acid. For example, substitutions are typically made within each of the following groups: (a) non-polar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine; (b) Polar neutral amino acids such as glycine, serine, threonine, cysteine, tyrosine, aspartic acid, and glutamic acid; (c) positively charged (basic) amino acids such as arginine, lysine, and histidine; (d) loading of aspartic acid and glutamic acid, etc. Electro (acidic) amino acids.

Modifications may be performed at any position on the amino acid sequence of the antibody, including the HVR, framework region, or constant region. In one embodiment, the disclosure provides an antibody derivative comprising the _VH and _VL HVR sequences of an exemplary antibody of the present disclosure, but comprising a framework sequence different from that of the exemplary antibody. Such framework sequences are available from public DNA databases containing germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kabat, EA, et al., Sequences of). Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991), Tomlin. -798 (1992); and Cox, J.P.L. et al., Eur. J. Immunol. 24: 827-836 (1994)). The framework sequences can be used in the construction of an antibody derivative, which framework sequences structurally similar to be used by exemplary antibodies of the present disclosure, for example, is used by the exemplary antibodies of the present disclosure, V _H 3-23 framework sequences and / or _VL λ3 or similar to λ1-13 framework sequences are included. For example, the HVR_H1, HVR_H2, and HVR_H3 sequences of an exemplary antibody, and the HVR_L1, HVR_L2, and HVR_L3 sequences have the same sequences found in the germline immunoglobulin gene from which the framework sequence is derived. Or the HVR sequence can be transplanted into a framework region containing one or more mutations compared to the germline sequence.

In certain embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an exemplary antibody of the disclosure. In one example, one or more HVRs from one or more exemplary human antibodies are combined with HVRs from antibodies from non-human animals such as mice or rats. In another example, all HVRs of chimeric antibodies are derived from one or more exemplary antibodies. In some specific embodiments, the chimeric antibody comprises one, two, or three HVRs derived from the heavy or light chain variable region of an exemplary antibody. Chimeric antibodies can be produced using conventional methods known in the art.

Another type of modification is to mutate amino acid residues within the HRV region of the _VH and / or _VL chain. Site-specific mutagenesis or PCR-mediated mutagenesis is performed to introduce the mutation (s) and the effect on antibody binding or other functional properties of interest is evaluated by in vitro or in vivo assays known in the art. it can. Typically, a conservative replacement is introduced. Mutations can be amino acid additions and / or deletions. In addition, typically no more than 1, 2, 3, 4, or 5 residues within the HVR region are altered. In some embodiments, the antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or the light chain HVR. In another embodiment, the amino acid substitution is to change one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. Cysteine can be standard or non-standard cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region as compared to the amino acid sequence of an exemplary antibody.

Modifications can also be made to framework residues within the _VH and / or _VL region. Typically, such framework variants are made to reduce the immunogenicity of the antibody. One approach is to "reverse" one or more framework residues to the corresponding germline sequence. Antibodies that have undergone a somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequence of the antibody with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline composition, somatic mutations can be "reverse-mutated" to germline sequences, for example by site-specific mutagenesis or PCR-mediated mutagenesis.

In addition, exemplary antibodies typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Modifications may be made within the Fc region of. In one example, the hinge region of CH1 is modified to vary in the number of cysteine residues in the hinge region, eg, increase or decrease. This approach is further described in US Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate light and heavy chain assembly, or to increase or decrease antibody stability. In other cases, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody.

In addition, the antibodies of the present disclosure can be modified to alter potential glycosylation sites or patterns according to routine experiments known in the art. In another aspect, the disclosure provides derivatives of the CD137 antibody of the present disclosure that contain at least one mutation in the variable region of the light or heavy chain that alters the pattern of glycosylation of the variable region. Such antibody derivatives may have increased affinity and / or modified specificity for binding to the antigen. Mutations add new glycosylation sites to the V region, reposition one or more glycosylation sites in the V region (s), or delete existing glycosylation sites in the V region. .. In one embodiment, the present disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site for asparagine in the heavy chain variable region and potential N-linked glycosylation of one heavy chain variable region. The site is removed. In another embodiment, the present disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site for asparagine in the heavy chain variable region and potential N-linked glycosylation in both heavy chain variable regions. The glycosylation site is removed. Methods of altering the glycosylation pattern of an antibody are known in the art and are described, for example, in US Pat. No. 6,933,368, the disclosure of which is incorporated herein by reference. Is done.

In another aspect, the disclosure provides an antibody derivative comprising the CD137 antibody described herein or an antigen-binding fragment thereof linked to an additional molecular entity. Examples of additional molecular entities include pharmaceuticals, peptides or proteins, detectors or labels, and antibodies.

In some embodiments, the antibody derivative comprises an antibody of the present disclosure linked to a pharmaceutical product. Examples of pharmaceuticals include cytotoxic agents or other cancer therapeutics, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytocaracin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corhitin, doxorubicin, daunorubicin, dihydroxyanthracycline, mitoxantrone, mitoxantrone. , Actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or relatives. Therapeutic agents also include, for example, metabolic antagonists (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, citaravin, 5-fluorouracil decarbazine), alkylating agents (eg, mechloretamine, thioepachlorambusyl, melphalan) , Carmustine (BSNU), and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (previously) Daunorubicin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthracycline (AMC)), and thread division inhibitors (eg, vincristine and vinblastine). Is done. Examples of radioisotopes that can be attached to an antibody for diagnostic or therapeutic use include, but are not limited to, iodine- ¹³¹ , indium- ¹¹¹ , yttrium- ⁹⁰ , and lutetium- ¹⁷⁷ . Methods of linking an antibody to a pharmaceutical product, such as using various linker techniques, are known in the art. Examples of linker types include hydrazone, thioether, esters, disulfides, and peptide-containing linkers. For more information on how to ligate a therapeutic agent to an antibody, see Saito et al. , Adv. Drag Deliv. Rev. 55: 199-215 (2003); Trail, et al. , Cancer Immunol. Immunother. 52: 328-337 (2003); Payne, Cancer Cell 3: 207-212 (2003); Allen, Nat. Rev. Cancer 2: 750-763 (2002); Pastan, I. et al. and Kreitman, Curr. Opin. Investig. Drugs 3: 1089-1091 (2002); Senter, P. et al. D. and Springer, C.I. J. (2001) Adv. Drag Deliv. Rev. See also 53: 247-264.

In certain embodiments, the antibody derivative is a CD137 antibody multimer, which is a multimeric form of a CD137 antibody, such as an antibody dimer, trimer, or higher multimer of a monomeric antibody. The individual monomers in the antibody multibody may be the same or different. In addition, the individual antibodies in the multibody may have the same or different binding specificities. Multimerization of the antibody can be achieved by the natural aggregation of the antibody. For example, some of the purified antibody preparations (eg, purified IgG4 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher antibody multimers. Alternatively, the antibody homodimer may be formed by a chemical binding technique known in the art, such as the use of a cross-linking agent. Suitable cross-linking agents include heterobifunctional (m-maleimidebenzoyl-N-hydroxysuccinimide ester, succinimidyl 4- (maleimidemethyl) cyclohexane-1-carboxy) having two distinct reactive groups separated by suitable spacers. Rate and N-succinimidyl S-acetylthio-acetate, etc.), or homobifunctional (such as dissuccinimidyl succinate). Such linkers are commercially available, for example, from Pierce Chemical Company, Rockford, IL. Antibodies can also be made to multimerize by recombinant DNA techniques known in the art.

In some embodiments, the antibodies of the present disclosure are multimeric antibodies (eg, bispecific antibodies). In some embodiments, the antibodies of the present disclosure are IgM antibodies and include, for example, an IgM Fc region (eg, a human IgM Fc region).

Examples of other antibody derivatives provided by the present disclosure include single chain antibodies, diabodies, domain antibodies, Nanobodies, and Unibodies. "Single-chain antibody" (scFv) consists of a single polypeptide chain comprising a V _L domain linked to a V _H domain, V _L and V _H domains to form monovalent molecules in pairs. Single chain antibodies can be prepared according to methods known in the art (eg, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883.). A "diabody" consists of two strands, each containing a heavy chain variable region connected to a light chain variable region on the same polypeptide chain connected by a short peptide linker, 2 on the same strand. The two regions are not paired with each other, but have domains complementary to the other strand, forming a bispecific molecule. Methods of preparing the diabody are known in the art (eg, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448, and Poljak R. J. et. al., (1994) Structure 2: 1121-1123). Domain antibodies (dAbs) are small functional binding units of an antibody that correspond to the variable region of either the heavy or light chain of the antibody. Domain antibodies are well expressed in bacterial, yeast and mammalian cell lines. Further details of the domain antibody and its production method are known in the art (eg, US Pat. No. 6,291,158; US Pat. No. 6,582,915; US Pat. No. 6,593,081). US Pat. No. 6,172,197; US Pat. No. 6,696,245; European Patent No. 0368684, and European Patent No. 06166640; WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, See WO04 / 003019 and WO03 / 002609). Nanobodies are derived from the heavy chains of antibodies. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, eg, US Pat. No. 6,765,087, US Pat. No. 6,838,254, WO 06/079372). The unibody consists of one light chain and one heavy chain of IgG4 antibody. Unibody can be made by removing the hinge region of the IgG4 antibody. Further details of the unibody and the method of preparing them can be found in WO2007 / 059782.

C. Recombinant Methods for Producing Nucleic Acids, Vectors, Host Cells, and CD137 Antibodies Another aspect of the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the binding molecule provided by the present disclosure. To do. The amino acid sequence encoded by the nucleotide sequence may be any portion of the antibody, such as a sequence containing HVRs, 1, 2, or 3 HVRs, variable regions of the heavy chain, variable regions of the light chain, or full length. It may be a heavy chain or a full length light chain. The nucleic acids of the present disclosure can be, for example, DNA or RNA and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

In some embodiments, the disclosure provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (1) of exemplary antibodies. Amino acid sequence of HVR_H3 or HVR_L3; (2) variable region of heavy chain or light chain of exemplary antibody; or (3) full length heavy chain or light chain of exemplary antibody.

In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 13-132, 253-612, 613-660, and 709-852. ..

In yet another embodiment, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 133-252 and 661-708.

The nucleic acids of the present disclosure can be obtained using any suitable molecular biology technique. In the case of antibodies expressed by hybridomas, the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), the nucleic acid encoding the antibody can be recovered from the library.

The isolated DNA encoding the V _H region is complete by operably linking the DNA encoding the V _H to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). Can be converted to long heavy chain genes. The sequences of human heavy chain constant region genes are known in the art (eg, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Technology Phase Humans (See No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but most preferably an IgG4 or IgG2 constant region without ADCC effect. The IgG4 constant region sequence can be any of a variety of alleles or alleles known to occur between different individuals. These allotypes represent naturally occurring amino acid substitutions in the IgG4 constant region. In the case of the Fab fragment heavy chain gene, the DNA encoding _VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the _VL region is converted to a fully long light chain gene by operably linking the _VL- encoding DNA to another DNA molecule encoding the light chain constant region, CL. Can be done. The sequence of the human light chain constant region gene is known in the art (eg, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Polymerase Technology of Humans (See No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the DNA fragment encoding the _{V H} and _{V L,} so that it can express the single-chain proteins consecutive _{which V H} and _{V L} sequences _{V L} and _{V H} regions joined by the flexible linker Is operably linked to, for example, another fragment encoding a flexible linker encoding amino acid sequence (Gly _4- Ser) ₃ (eg, Bird et al., Science 242: 423-426 (1988); Huston). et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and McCafferty et al., Nature 348: 552-554 (1990)).

The disclosure further provides a vector containing the nucleic acid molecules provided by the disclosure. Nucleic acid molecules are light chain or heavy chains (such as CDR or HVR), full length light chains or heavy chains, heavy chains or polypeptides containing part or full length of light chains, or amino acids of antibody derivatives or antigen binding fragments. You may code an array. In some embodiments, the vector is an expression vector useful for the expression of a binding molecule, such as an antibody or antigen-binding fragment thereof. In some embodiments, vectors are provided, the first vector comprising a polynucleotide sequence encoding the heavy chain variable region described herein, and the second vector containing the light chain variable region described herein. Contains the polynucleotide sequence to encode. In some embodiments, the single vector comprises a polynucleotide encoding a heavy chain variable region described herein and a light chain variable region described herein.

To express the binding molecules of the present disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector so that the DNA molecules are operably linked to transcription and translation control sequences. To do. In this context, the term "operably linked" refers to the binding of an antibody gene to a vector such that the transcription and translation control sequences within the vector perform their intended function of regulating the transcription and translation of DNA molecules. Means to be done. The expression vector and expression control sequence are selected to be compatible with the expression host cell used. The light chain gene of the antibody and the heavy chain gene of the antibody can be inserted into separate vectors, more typically both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (eg, ligation of the antibody gene fragment and complementary restriction sites on the vector, or homologous recombination-based DNA ligation). Light and heavy chain variable regions of the antibodies described herein is inserted into an expression vector that already encoding heavy chain constant and light chain constant regions of the desired isotype and subclass, V _H segment in the vector C _H segment (s) operably linked to, by such V _L segment is operatively linked to the C _L segment within the vector, to create a full-length antibody genes any antibody isotype and subclass Can be used for. Further, or alternative, the recombinant expression vector can encode a signal peptide that stimulates the secretion of antibody chains from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain gene, the expression vectors of the present disclosure typically carry regulatory sequences that control the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such control sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, California. (1990)). It will be appreciated by those skilled in the art that the design of an expression vector, including the selection of control sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Examples of regulatory sequences for mammalian host cell expression include cytomegalovirus (CMV), Simianvirus 40 (SV40), adenovirus (eg, adenovirus major late promoter (AdMLP) and polyoma-derived promoters and / or enhancers. , Includes viral elements that induce high levels of protein expression in mammalian cells. Alternatively, nonviral regulatory sequences such as the ubiquitin promoter or β-globin promoter may be used. In addition, regulatory elements may be used. Consists of sequences from different sources, such as the SR promoter system containing sequences from the SV40 early promoter and long terminal repeats of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8). : 466-472).

In addition to the antibody chain gene and control sequence, the expression vector may carry additional sequences such as sequences that regulate the replication of the vector in the host cell (eg, the origin of replication) and selectable marker genes. Selectable marker genes facilitate selection of the host cell into which the vector has been introduced (eg, US Pat. No. 4,399,216, US Pat. No. 4,634,665, all by Axel et al., And US Pat. No. 5,179,017). For example, typically selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

For light and heavy chain expression, the expression vector (s) encoding the heavy and light chains are transfected into the host cell by any suitable technique. The various forms of the term "transfection" are a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE. -Intended to include dextran transfection and the like. Although it is possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and typically mammalian host cells, is most typical. ..

The disclosure further provides host cells containing the nucleic acid molecules provided by the disclosure. The host cell can be virtually any cell for which an expression vector is available. It may be, for example, a higher eukaryotic host cell such as a mammalian cell, a lower eukaryotic host cell such as a yeast cell, or a prokaryotic cell such as a bacterial cell. Introduction of recombinant nucleic acid constructs into host cells can be achieved by calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

Prokaryotic hosts suitable for transformation include E. coli. Includes E. coli, Bacillus subtilis, Salmonella enterica, and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus.

Mammalian host cells for expressing the binding molecules of the present disclosure are described, for example, in Chinese Hamster Ovary (CHO) cells (eg, Kaufman and Sharp, J. Mol. Biol. 159: 601-621 (1982)). As described in Urlaub and Sharp, Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980), including dhfr-CHO cells), NS0 myeloma cells, COS, used with DHFR. Includes cells and Sp2 cells. In particular, another expression system for use in NS0 myeloma or CHO cells is the GS (glutamine synthetase) gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. When an expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is used in the host for a period sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the medium in which they grow. Produced by culturing cells. Antibodies can be recovered from the medium using appropriate protein purification methods.

D. Composition In another aspect, the present disclosure provides a composition comprising a binding molecule provided by the present disclosure. In one embodiment, the composition is a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

In some embodiments, the disclosure provides a composition comprising an antibody or antigen-binding fragment thereof provided by the present disclosure and a pharmaceutically acceptable carrier, wherein the antibody is disclosed herein. The composition comprises a variable domain comprising the HVR amino acid sequence to be: about 11%, 10%, 8%, 5%, 3%, or 2% or less of the antibody, or antigen binding moiety. It is glycosylated with asparagine of the amino acid sequence as compared to the total amount of antibody or antigen-binding portion thereof present in the composition. In another embodiment, the composition comprises at least about 2% of the antibody or antigen-binding moiety of the amino acid sequence as compared to the total amount of the antibody or antigen-binding moiety present in the composition. It is glycosylated with asparagine.

The term "pharmaceutically acceptable carrier" refers to any Inactive substance suitable for use in a formulation for delivery of a binding molecule. Carriers include anti-adhesion agents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption retarders, wetting agents, emulsifiers , Buffer solution, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (olive oil, etc.), saline, buffers, buffered saline, etc. Includes isotonic agents such as sugar, polyhydric alcohol, sorbitol, sodium chloride.

The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (eg, injectable and injectable solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of composition suitable for delivering a binding molecule is a sterile solution, such as a solution, suspension, or dispersion for injection or infusion. The sterile solution can be prepared by mixing the required amount of antibody with a suitable carrier and then performing sterile microfiltration. Generally, the dispersion is prepared by mixing the antibody with a sterile vehicle containing a basic dispersion medium and other carriers. For sterile powders for the preparation of sterile liquids, preparation methods include vacuum drying and lyophilization (lyophilization), with the active ingredient and any additional desired ingredients from the previously sterile filtered solution. Powder is obtained. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

The relative amount of binding molecule contained in the composition will vary depending on many factors such as the particular binding molecule and carrier used, dosage form, and desired release and pharmacodynamic properties. The amount of binding molecule in a single dosage form will generally be the amount that produces a therapeutic effect, but may be less. Generally, this amount will range from about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of the total weight of the dosage form.

In addition to the binding molecule, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described herein below. The appropriate amount of additional therapeutic agent to be included in the composition can be readily selected by one of ordinary skill in the art, such as the particular agent and carrier used, dosage form, and desired release and pharmacodynamic properties. It will vary depending on many factors. The amount of additional therapeutic agent in a single dosage form will generally be the amount of agent producing a therapeutic effect, but may also be a smaller amount.

E. Use of Binding Molecules and Pharmaceutical Compositions The binding molecules and pharmaceutical compositions provided by the present disclosure are the regulation of immune response, the treatment of cancer, the enhancement of other cancer treatments, the enhancement of vaccine efficacy, or autoimmune It is useful for therapeutic, diagnostic, or other purposes, such as the treatment of immune disorders. Thus, in other embodiments, the present disclosure provides a method of using a binding molecule or pharmaceutical composition. In one aspect, the disclosure provides a method of treating a mammalian disorder, comprising administering to a mammal in need of treatment a therapeutically effective amount of a binding molecule provided by the present disclosure. The binding molecule can be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In some embodiments, the mammal is a human.

In some embodiments, the disorder is cancer. Various cancers associated with CD137, whether malignant or benign, primary or secondary, can be treated or prevented by the methods provided in this disclosure. Examples of such cancers include bronchial cancers (eg, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, sarcoma error (non-cancerous). , Pulmonary cancer such as sarcoma (cancerous); Heart cancer such as mucinoma, fibroma, rhizome myoma; osteochondroma, chondroma, chondrocyte blastoma, cartilage mucinous fibroma, osteoarthritis, giant cell tumor , Chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing tumor (Ewing sarcoma), and reticular sarcoma; glioma (eg, polymorphic glioblastoma) ), Malignant stellate cell tumor, stellate cell tumor, oligodendroglioma, medullary blastoma, sarcoma, nerve sheath tumor, lining tumor, sarcoma, pituitary adenoma, pineapple tumor, osteoma, blood vessel Brain cancers such as blastoma, cranopharyngeal tumor, spinal cord tumor, germ cell tumor, malformation, cutaneous sarcoma, and hemangiomas; smooth myoma, epidermoid cancer, adenocarcinoma, smooth sarcoma, gastric adenocarcinoma, intestinal lipoma, Gastrointestinal cancers such as enteric nerve fibroma, intestinal fibroma, colon polyp, colonic rectal cancer; hepatocellular adenomas, hemangiomas, hepatocellular carcinomas, fibrous layer cancers, bile duct cancers, hepatoblastomas, angiosarcomas, etc. Liver cancer; renal adenocarcinoma, renal cell carcinoma, adrenoma, renal cancer such as transitional epithelial cancer of the renal pelvis; bladder cancer; acute lymphocytic (lymphocytic) leukemia, acute myeloid (myelocytic, myeloid, bone marrow) Sprouting, myeloid monocytic) leukemia, chronic lymphocytic leukemia (eg, Cesarly syndrome and hair cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) leukemia, hodgkin lymphoma, non-hodgkin lymphoma , B-cell lymphoma, fungal sarcoma, and hematological malignancies such as myeloproliferative disorders (including myeloproliferative disorders such as true erythrocytosis, myelodystrophy, thrombosis, chronic myeloid leukemia); basal cell carcinoma Skin cancers such as squamous cell carcinoma, melanoma, caposarcoma, Paget's disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma; benign prostate hyperplasia, prostate cancer, and testicular cancer Male reproductive system cancers (eg, sarcoma, malforma, embryonic cancer, chorionic villus cancer); breast cancer; uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian cancer) ), Cancers of the female reproductive system such as genital cancer, vaginal cancer, oviduct cancer, and cystic sarcoma; thyroid cancer (including papillary cancer, follicular cancer, undifferentiated cancer, or medullary cancer); brown cell tumor ( Adrenal); non-cancerous growth of the parathyroid; pancreatic cancer; hematological cancers such as leukemia, sarcoma, non-hodgkin lymphoma, and hodgkin lymphoma.

In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with binding molecules include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The binding molecule can also be used to treat inflammation (such as allergic asthma) and chronic graft-versus-host disease.

In another aspect, the disclosure provides a method of enhancing a mammalian immune response, comprising administering to the mammal a therapeutically effective amount of a binding molecule provided by the present disclosure. In some embodiments, the binding molecule is a CD137 antibody or antigen-binding fragment thereof and the mammal is a human. In a further embodiment, the binding molecule is a CD137 agonist antibody or antigen binding fragment thereof. The term "enhancing the immune response" or its grammatical changes means stimulating, inducing, increasing, ameliorating, or enhancing the response of the mammalian immune system. The immune response may be a cellular response (ie, cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (ie, antibody-mediated response), and may be a primary or secondary immune response. .. Examples of enhanced immune response include increased CD4 + helper T cell activity and production of cytolytic T cells. Enhanced immune response includes cytotoxic T lymphocyte assay, cytokine release (eg, IL-2 production), tumor regression, tumor-bearing animal survival, antibody production, immune cell proliferation, expression of cell surface markers, cells It can be evaluated using a number of in vitro or in vivo measurements known to those of skill in the art, including but not limited to cytotoxicity. Typically, the methods of the present disclosure enhance an immune response by a mammal when compared to an immune response by an untreated mammal or an untreated mammal using the claims method. .. In one embodiment, the binding molecule is used to enhance the human immune response to microbial pathogens (such as viruses). In another embodiment, the binding molecule is used to enhance the human immune response to the vaccine. The binding molecule can be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In one embodiment, the method enhances a cell-mediated immune response, particularly a cytotoxic T cell response. In another embodiment, the cell-mediated immune response is a T-helper cell response. In yet another embodiment, the immune response is cytokine production, especially IL-2 production. Bound molecules can be used to enhance a human's immune response to a microbial pathogen (such as a virus), or vaccine. The binding molecule can be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist.

In practicing the method of treatment, the binding molecule may be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the disclosure provides a combination therapy comprising a binding molecule in combination with one or more additional therapies or therapeutic agents for separate, sequential, or co-administration. The term "additional therapy" refers to a therapy that does not use the binding molecule provided by the present disclosure as a therapeutic agent. The term "additional therapeutic agent" refers to any therapeutic agent other than the binding molecule provided by the present disclosure. In one particular aspect, the disclosure treats mammalian cancer, comprising administering to the mammal a therapeutically effective amount of a binding molecule provided by the present disclosure in combination with one or more additional therapeutic agents. Provide a combination therapy to do. In a further embodiment, the mammal is a human.

A wide variety of cancer therapeutic agents can be used in combination with the binding molecules provided by the present disclosure. Those skilled in the art will be able to recognize the existence and development of other cancer therapies that can be used in combination with the methods and binding molecules of the present disclosure and are not limited to the forms of therapy described herein. Let's do it. Examples of additional therapeutic agent categories that can be used in combination therapies to treat cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents.

The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause the death of cancer cells or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO2006 / 129163 and US20060153388, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) chlorambusyl (leukelan), mucyclophosphamide (citoxane), ifofamide (IFEX), mechloretamine hydrochloride (mustalgen), thiotepa (thioplex), streptozotocin ( Alkylating agents such as Zanosar), carmustin (BICNU, gliadelwaf), romustin (CEENU), and dacarbazine (DTIC-DOME); (2) doxorubicin (adriamycin), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (serubicin) ), Nemorphicin, Idarubicin (IDAMYCIN PFS, ZAVEDOS), Mitoxantron (DHAD, NOVANTRONE), Dactinomycin (Actinomycin D, Cosmegen), Prikamycin (Mitracin), Mitomycin (Mutamycin), and Breonomycin (Brenoxane). Alkaloids or plant vinca alkaloids, including cytotoxic antibiotics, binorerbin tartrate (navelbin), vinblastin (VELBAN), vincristin (oncobin), and bindesin (ELDISINE); (3) capecitabin (Xeloda), citarabin (CYTOSAR-U) , Fludarabin (Fludara), Gemcitabine (Gemzar), Hydroxyurea (HYDRA), Methotrexate (FOLEX, MEXATE, TREXALL), Nerarabin (Alanon), Trimetrexate (NEUTREXIN), and Methotrexate (Alimta) 4) 5-Fluorouracil (5-FU); pyrimidin antagonists such as capecitabin (Xeloda), larcitrexed (TOMUDEX), tegafururacil (UFTORAL), and gemcitabine (gemzar); (5) docetaxel (taxotere), paclitaxel (taxol) Taxan such as (6) Platinum preparations such as cisplatin (platinol), carboplatin (paraplatin), oxaliplatin (eroxatin); (7) irinotecan (CAMPTOSAR), topotecan (hycamtin), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), teniposide Topoisomerase inhibitors such as (VUMON); (8) Epis such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR) Podophilotoxin (podophylrotoxin derivative); (9) Folic acid derivative such as leucovorin (wellcobolin); (10) Nitrosurea such as carmustin (BiCNU), romustin (CeeNU); (11) Gefitinib (Iressa), Elrotinib (Tarceva), bortezomib (Velcade), imatinib mesylate (Gleevec), gefitinib, rapatinib, sorafenib, salidamide, snitinib (satin), axitinib, rituximab (rituxan, mabusera), trussimab (herceptin), trussemab (herceptin) Epithelial growth factor receptors (EGFR), such as bevasizumab (Avastin), ranibizumab (Lucentis), lym-1 (Oncolim), antibodies against the insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596. ), Vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocellular growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR). (12) Angiogenesis inhibitors such as bevasizumab (Avastin), slamin (GERMANIN), angiostatin, SU5416, salidamide, and matrix metalloproteinase inhibitors (such as batimastat and marimasut), and WO200205516. What is; as well as (13) proteasome inhibitors such as bortezomib (Velcade).

The term "immunotherapeutic agent" refers to a chemical or biological substance that can enhance the immune response of a mammal. Examples of immunotherapeutic agents include: sterilized bovine tuberculosis (BCG); cytokines such as interferon; MyVax personalized immunotherapy, Onevax-P, oncophages, GRNVAC1, Fabld, Provenge, GVAX, Lovaxin C, Biovax ID. , GMXX, NeuVax and other vaccines; as well as alemtuzumab (campus), bebashizumab (avastin), cetuximab (erbitux), gemtuzumab ozogamicin (myrotag), ibritsumumabuchiuxetan (zevalin), panitumumab (bektimumab) (Rituxan, Mabusera), trastuzumab (Herceptin), tocitumomab (Bexal), ipilimumab (Yervoy), Tremerimumab, CAT-3888, agonist antibodies against OX40 receptors (such as those disclosed in WO2009 / 079335), against CD40 receptors Antibodies such as agonist antibodies (such as those disclosed in WO2003 / 040170) and TLR-9 agonists (such as those disclosed in WO2003 / 015711, WO2004 / 06805, and WO2009 / 022215).

The term "hormonal therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones or inhibits or offsets the effects of hormones on the growth and / or survival of cancerous cells. Examples of such agents suitable for the methods herein include those disclosed in US20070117809. Examples of specific hormone therapies include tamoxifen (Nolvadex), toremifene (Fairston), fulvestrant (Fesolodex), anastrozole (Arimidex), exemestane (Aromasin), letrozole (Femara), and meacetic acid. Includes guest rolls (MEGACE), goserelin (Zoladex), and leuprolide (Lupron). The binding molecules of the present disclosure include (1) surgical methods to remove all or part of the organs or glands involved in the production of hormones such as the ovaries, testicles, adrenal glands, and pituitary glands, and (2) the patient's organs or It can also be used in combination with non-drug hormone therapy, such as radiotherapy, where the glands are exposed to sufficient amounts of radiation to inhibit or eliminate the production of target hormones.

Combination therapies to treat cancer also include a combination of binding molecules and surgery to remove the tumor. The binding molecule can be administered to the mammal before, during, or after surgery.

Combination therapies for treating cancer can also be combined with bound molecules and radiation therapies such as ionizing (electromagnetic) radiation therapy (eg, X-rays or gamma rays) and particle beam therapy (eg, high linear energy radiation). Included. The radiation source can be outside or inside the mammal. The binding molecule can be administered to the mammal before, during, or after radiation therapy.

The binding molecules and compositions provided by the present disclosure can be administered via any suitable enteric or parenteral route of administration. The term "enteric route" of administration refers to administration via any part of the gastrointestinal tract. Examples of transintestinal routes include oral, mucosal, buccal, rectal, or intragastric routes. The "parenteral route" of administration refers to a route of administration other than the intestinal route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intrasternal, intraorbital, intracardiac, transtracheal, intra-articular, subcapsular. Includes subepithelial, intraspinal, epidural and intrasternal, subcutaneous, or topical administration. The antibodies and compositions of the present disclosure can be administered using any suitable method such as ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. .. Appropriate routes of administration and methods of administration include the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the disorder being treated, the particular site of action, and the condition of the patient. It can vary depending on many factors such as, and can be easily selected by those skilled in the art.

The term "therapeutically effective amount" of a binding molecule refers to an amount effective for the intended therapeutic purpose. For example, in the context of enhancing an immune response, a "therapeutically effective amount" is any amount that is effective in stimulating, inducing, increasing, ameliorating, or enhancing any response of the mammalian immune system. In the context of treating a disease, a "therapeutically effective amount" is any amount sufficient to cause any desired or beneficial effect on the mammal being treated. Specifically, examples of desirable or beneficial effects in the treatment of cancer are associated with inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, cancer. Includes pain relief or improved mammalian survival. The therapeutically effective amount of the CD137 antibody is typically in the range of about 0.001 to about 500 mg / kg, more generally about 0.01 to about 100 mg / kg, based on the body weight of the mammal. For example, the amount can be about 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, or 100 mg / kg based on the body weight of the mammal. In some embodiments, the therapeutically effective amount of CD137 antibody is in the range of about 0.01-30 mg / kg based on the body weight of the mammal. In some other embodiments, the therapeutically effective amount of the CD137 antibody ranges from about 0.05 to 15 mg / kg based on the body weight of the mammal. The exact dose level administered depends on the type and severity of the disorder being treated, the specific binding molecule used, the route of administration, the time of administration, the duration of treatment, the specific additional therapy used, and the patient being treated. It can be easily determined by those skilled in the art, depending on age, gender, weight, condition, overall health, and many factors of past medical history, as well as similar factors well known in the medical field.

The binding molecule or composition is usually administered multiple times. The interval between single doses can be, for example, weekly, monthly, every three months, or yearly. An exemplary treatment plan is once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. With multiple doses. A typical dosing regimen for the CD137 antibody includes 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration and uses one of the following dosing schedules. (I) 6 doses every 4 weeks, then every 3 months; (ii) every 3 weeks; (iii) 3 mg / kg body weight once, then every 3 weeks 1 mg / kg body weight.

The present disclosure will be more fully understood by reference to the examples below. However, the examples should not be construed as limiting the scope of this disclosure. The examples and embodiments described herein are for purposes of illustration only, and various modifications or modifications made in consideration thereof have been suggested to those skilled in the art, which are the scope of the present application and the accompanying claims. It will be understood that it is included in the purpose and scope of. All figures and all references, patents, and published patent applications cited throughout this disclosure are expressly incorporated herein by reference in their entirety.

Example 1
Generation of a primary Fab that specifically binds to human CD137 A proprietary phagemid library (incorporated herein by reference, referred to herein as Agent Reference No. 69540-2000140, filed at the same time as this specification, "Dynamic Human Antibody Light Chain". See a PCT international application entitled "Libraries"; entitled "Dynamic Human Hairy Chain Antibodies", which is incorporated herein by reference and is filed at the same time as this specification as agent reference number 69540-2000240. PCT international application (see also) was used to pan the human CD137 antigen. A total of 3 or 4 pannings were performed. After the final round of panning, a single colony supernatant ELISA was performed to identify primary hits that specifically recognize human CD137. The primary hit was defined as having an ELISA signal at least twice the background. They were sequenced, unique clones were expressed and purified for affinity measurements by ForteBio and Biacore. The list was narrowed down to 124 with Fabs containing both Elisa positive hits and unique sequences. K _D response signal R> ^0.1, according to the criteria of the R 2> 0.9, and affinity _K D <100 nM, list was narrowed further 60 hits (Table 1a). Twenty-four of them were then converted to IgG for detailed biophysical and functional characterization (Table 1b).

The Fab corresponding to the unique hit is E.I. It was expressed in E. coli and purified. Their affinity for human CD137 was measured by ForteBio Octet RED96 Systems. Briefly, CD137-hisFc fusion protein (Sino Biological # Cat 10041-H03H) was captured using AHC sensors (anti-human IgG FcCapture Dip and Read Biosensor) and kinetic buffer (10 mM HEPES, 150 mM NaCl, 3 mM). It was immersed in a well containing purified Fab diluted to 5-10 μg / ml with HEPES, 0.005% v / v Surfactant P20, pH 7.4). The acquired ForteBio data was processed by data acquisition software 7.1 and the dynamic data was fitted to a 1: 1 Langmuir coupling model. Affinity and kinetic parameters (subtracted background) are listed in Table 1a. The affinity of their corresponding IgG for human CD137 was measured with Biacore and is shown in Table 1b.
Table 1a Affinity of the selected Fab for human CD137 and the corresponding amino acid sequence (SEQ ID NO:)

Corresponding DNA sequences encoding the amino acid sequences of SEQ ID NOs: 13-132 can be found in SEQ ID NOs: 133-252, respectively. The HVR_H1 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 253 to 312, respectively. The HVR_H2 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 313-372, respectively. The HVR_H3 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 373-432, respectively. The HVR_L1 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 433-492, respectively. The HVR_L2 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 493 to 552, respectively. The HVR_L3 amino acid sequences of all Fab hits shown in Table 1a can be found in SEQ ID NOs: 553 to 612, respectively (see also Table 1c).
Table 1b Affinity of Fab and corresponding IgG for human CD137

Corresponding DNA sequences encoding the amino acid sequences of SEQ ID NOs: 613-660 can be found in SEQ ID NOs: 661-708, respectively. The HVR_H1 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 709-732, respectively. The HVR_H2 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 733-756, respectively. The HVR_H3 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 757-780, respectively. The HVR_L1 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 781-804, respectively. The HVR_L2 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 805-828, respectively. The HVR_L3 amino acid sequences of all the IgG sequences shown in Table 1b can be found in SEQ ID NOs: 829-852, respectively.
Table 1c: Fab CDR sequence

Example 2
Selection of Fab Hits Cross-Reactive with Mouse CD137 Fab Hit Species Cross-Reactivity was determined using ELISA. Briefly, 200 μL of 5 μg / mL m of anti-human IgG (Fab-specific) (Sigma # I5260) was coated on a Maxisorpμ plate (Thermo Scientific 446469) overnight at 4 ° C. After blocking, 100 μL of Fab5310 (5 μg / mL), 5351 (2.8 μg / mL) and 5365 (5 μg / mL) were added and incubated for 1 hour. After washing 3 times, a serial dilution of human or mouse CD137 antigen fused with a human FC fragment was added and incubated for 1 hour. After washing, HRP-labeled goat anti-human FC was diluted 1: 2000 with PBS, added to each well and incubated for 1 hour. The plates were washed 3 times and incubated with TMB substrate for 20 minutes at room temperature. After the reaction was stopped, the absorbance at 450 nm was measured. The results are shown in FIG. 1b, and the lower panel shows that Fab 5310 and 5365 bind to both human and mouse CD137, whereas Fab5351 binds to human CD137 but not mouse CD137.

Example 3
IgG conversion and expression: AG10048, AG10045, and AG10131
The heavy and light chains of Fab5310, 5351, and 5365 were individually cloned into an IgG4 isotype containing the S241P mutation of the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific). The heavy and light chains of the two reference antibodies were also cloned into the IgG4 and IgG2 isotypes of pCDNA 3.3, respectively.

A heavy chain variable region that is used in reference antibody AC1097 comprises the sequence IbuikyuerubuikyuesujieiibuikeikeiPijiiesueruaruaiesushikeijiesujiwaiesuefuesutiwaidaburyuaiesudaburyubuiarukyuemuPijikeijieruidaburyuemujikeiaiwaiPijidiesuwaitienuwaiesuPiesuefukyujikyubuitiaiesueidikeiesuaiesutieiwaierukyudaburyuesuesuerukeieiesuditiAMYYCARGYGIFDYWGQGTLVTVSS (SEQ ID NO: 862), a light chain variable region that is used in reference antibody AC1097 contained the sequence EsuwaiierutikyuPiPiesubuiesubuiesuPijikyutieiesuaitishiesujidienuaijidikyuwaieieichidaburyuwaikyukyukeiPijikyuesuPibuierubuiaiwaikyudikeienuaruPiesujiaiPiiaruefuesujiesuenuesujienutieitierutiaiesujitikyueiemudiieiDYYCATYTGFGSLAVFGGGTKLTVL (SEQ ID NO: 863). A heavy chain variable region that is used in reference antibody AC1121 comprises the sequence KyubuikyuerukyukyudaburyujieijieruerukeiPiesuitieruesuerutishieibuiwaijijiesuefuesujiwaiwaidaburyuesudaburyuaiarukyuesuPiikeijieruidaburyuaijiiaienueichijijiwaibuitiwaienuPiesueruiesuarubuitiaiesubuiditiesukeienukyuefuesuerukeieruesuesubuitieieiditieibuiwaiwaishieiRDYGPGNYDWYFDLWGRGTLVTVSS (SEQ ID NO: 864), a light chain variable region that is used in reference antibody AC1121 contained the sequence IaibuierutikyuesuPieitieruesueruesuPijiiarueitieruesushiarueiesukyuesubuiesuesuwaierueidaburyuwaikyukyukeiPijikyueiPiaruerueruaiwaidieiesuenuarueitijiaiPieiaruefuesujiesujiesujitidiefutierutiaiesuesueruiPiidiefueiVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 865). The IgG used herein is shown in Table 2.
Table 2: List of IgG

A pair of plasmids was transiently transfected into HEK293F cells according to the manufacturer's instructions. The supernatant was collected, clarified by centrifugation and filtration, and IgG was purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The protein was eluted and neutralized, and the buffer was replaced with PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). Protein concentration was determined by UV spectrophotometric method and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

Example 4
Binding Affinity of Human, Monkey, and Mouse to CD137 The binding affinity of IgG for human, monkey, and mouse CD137 was measured by BIAcore, ELISA, and flow cytometry. The results are summarized in Table 3.
Table 3 Binding affinity of antibodies to CD137 in humans, monkeys and mice

4a. Measurement of binding affinity and kinetics by SPR According to manufacturer's guidelines, surface plasmon resonance (SPR) analysis using a Biacore ™ T200 instrument (Biacore AB, Uppsala, Sweden) was performed on human, monkey, and mouse CD137 proteins. The binding affinity and kinetics of the antibody against was investigated. The anti-human IgG (Fc) antibody of the Human Antibody Capture kit (GE BR-1008-39) has its amine group carboxylated to the sensor chip according to the instruction manual of the Amine Coupling kit (GE Biacore # BR-1000-50). It was fixed to the CM5 chip by coupling to the surface. Immobilized anti-human IgG (Fc) antibodies were used to capture AG10048, AG10059, AG10131, AC1121, and AC1097. Finally, human CD137-His6 (Sino Biological # 10041-H08H) at a concentration of 6 (3.13, 6.25, 12.5, 25, 50, 100) (nM) (diluted with running buffer) The injection was performed at a flow rate of 30 μI / min for 300 seconds, and the dissociation time was 300 seconds. The running buffer used was 1 × HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, pH 7.4 at 25 ° C.). For "background" subtraction, blank flow cells containing no immobilized protein were used to perform the corresponding controls in each case. According to the manufacturer's guidelines, the Biacore T200 evaluation software (Biacore AB, Uppsala, Sweden) was used to fit the binding and dissociation curves to the 1: 1 Langmuir binding model. As shown in Table 3, all antibodies bind to human CD137. AG10048 and AG10059 show higher affinity than both reference antibodies. See AC1121 All antibodies bind monkey CD137 except mAb. Only AG10048 and AG10131 bind to mouse and rat CD137. AG10048 has a higher affinity (15.2 nM) than AG10131 (64.5 nM).

4b. Measurement of Binding Affinity to Soluble CD137 Using ELISA Assay A serial dilution of human, monkey, or mouse CD137 fused with a human FC fragment was prepared and used to coat an ELISA plate at 37 ° C. for 1 hour. .. After blocking, 100 μL IgG (5 μg / mL) was added and incubated at 37 ° C. for 1 hour. The plates were washed 3 times and then incubated with HRP-conjugated protein L (1: 2000 dilution) at 37 ° C. for 1 hour. The plates were washed 3 times again and incubated with TMB substrate for 20 minutes at room temperature. After the reaction was stopped, the absorbance at 450 nm was measured. Data were analyzed by Graphpad Prism 6 with non-linear fitting. As shown in FIG. 2, all antibodies bind to human CD137 (FC fusion protein) with similar sub-nM affinity. Except for the AC1121 reference mAb, all antibodies bind monkey CD137 with similar sub-nM affinity. Consistent with the Biacore results, only AG10048 and AG10131 bind to mouse CD137. AG10048 has a higher affinity (0.3 nM) than AG10131 (23.9 nM).

4c. Measurement of Binding Affinity to CD137 Overexpressed on Cell Surface by Flow Cytometry Antibody affinity was also evaluated for human, monkey, and mouse CD137 transiently expressed on the surface of HEK293F cells. .. Briefly, HEK293F cells were transfected with a plasmid expressing full-length human, monkey, or mouse CD137 from a bicistron IRES vector and transfected cells were identified using EGFP. After 48 hours, the transfected cells were harvested and washed once with cold FACS buffer (PBS supplemented with 1% BSA). The cells were then incubated with various IgGs (100 nM each) for 1 hour on ice, washed twice with pre-cooled FACS buffer and 30 minutes on ice with AlexaFlower® 647-conjugated mouse anti-human FC antibody. Incubated. Cells were washed once prior to analysis by flow cytometry (Beckman® CytoFlex). As shown in FIG. 3a, all antibodies bind to human CD137 expressed on the cell surface with low nM affinity. AG10048, AG10059, and AG10131 are slightly superior to both reference antibodies. With the exception of the AC1121 reference mAb, all antibodies bind monkey CD137 with low nM affinity, and AG10058, AG10059, and AG10131 are slightly superior to the AC1097 reference antibody. Consistent with the results of Biacore and ELISA, only AG10048 and AG10131 bind to mouse and rat CD137. AG10048 has a higher affinity for mouse CD137 than AG10131. In addition, AG10048 and AG10131 (100 nM each) also bind to rat and dog CD137 overexpressed on the HEK293F cell surface (FIG. 3b).

4d. Binding of IgG to activated human, monkey, mouse, and rat T cells.
Species cross-reactivity of exemplary antibodies was further confirmed using human, monkey, mouse, and rat PMA and ionomycin-stimulated PBMCs or T cells. Human and cynomolgus monkey PBMCs were isolated by Ficoll density gradient centrifugation. Briefly, freshly collected whole blood from a healthy donor or cynomolgus monkey was diluted with an equal volume of PBS and carefully loaded onto the top of Histopaque 1077 (14 ml in a 50 ml centrifuge tube). Brake off and centrifuge at 1,200 xg for 30 minutes at room temperature. After centrifugation, carefully pipette the upper layer within 0.5 cm of the opaque interface containing mononuclear cells. Discard the upper layer. Carefully pipette the opaque interface (approximately 3-5 ml) into a clean 50 ml conical centrifuge tube. The cells are washed with 20 ml PBS and centrifuged at 400 xg for 5 minutes to collect the cells and resuspend the cells in 20 ml PBS. The cells are counted on a hemocytometer, centrifuged at 400 xg for 5 minutes, and the cells are collected again. Mouse or rat spleen cells were isolated by passing the spleen through a 45 μm cell strainer attached to a 50 mL conical tube to obtain a single cell suspension and washing the cells through the strainer with PBS. Centrifuge at 1600 rpm for 5 minutes and discard the supernatant. Resuspend the cell pellet in 2 ml of erythrocyte lysate for 2 minutes. Add more than 10 times the amount of PBS and centrifuge at 1600 rpm for 5 minutes to collect cells. Discard the supernatant and resuspend splenocytes in RPMI 1640/10% FBS. Pan-T cells are enriched from PBMCs (humans / monkeys) or splenocytes (mouse / rats) by negative selection with magnetic beads from a commercial kit (Semcell Technologies) specific for humans, monkeys, mice, and rats, respectively. It was. Activation of human / monkey PBMC or mouse / rat splenocytes was performed by incubating the cells with 50 ng / ml PMA and 1 μM ionomycin overnight at 37 ° C. and 5% CO2.

Washed with activated cells (about 2 × 10 ⁵ cells / tube) were cooled in advance staining buffer (PBS supplemented with 2% FBS), 1 h on ice with 100nM of test antibody. The cells were then washed twice with 1 mL staining buffer and resuspended in 100 μL staining buffer containing AlexaFluor® 647-conjugated mouse anti-human FC antibody and species-specific T cell marker antibody. did. T cell marker antibodies such as: CD3, CD4, or CD8 were used. After incubating for 30 minutes in the dark, cells were washed twice with staining buffer. Finally, cells were resuspended in 300 μL of staining buffer and analyzed with Beckman CytoFlex. Data analysis was performed using Flowjo 10 software. As shown in FIG. 4a, all antibodies tested bind to both activated human and monkey T cells, but not naive human T cells. The ability of AG10131 to bind to activated mouse and rat T cells was further evaluated (Fig. 4b). AG10131 binds to both activated mouse and rat T cells.

In summary, the AG10048 and AG10131 antibodies show higher affinity for human and monkey CD137. They exhibit a wide range of cross-reactivity for AG10131, including humans, cynomolgus monkeys, mice, rats, and dogs, whereas for AG10048, they include humans, cynomolgus monkeys, mice, and dogs and are effective in vivo in mouse syngeneic models. Allows rapid assessment of sex.

Example 5
Antibody Binding Selectivity to CD137 The antibody selectivity to CD137 was assessed using flow cytometric analysis of its ability to bind to members of the TNFR superfamily. TNFRSF receptors, including CD137, OX40, CD40, GITR, and CD27, were transiently overexpressed on the surface of HEK293F cells. Transfected cells were washed with pre-cooled staining buffer (PBS supplemented with 2% FBS) and then incubated on ice for 1 hour with 100 nM test antibody. Cells were washed twice with staining buffer, AlexaFluor® 647-conjugated mouse anti-human FC antibody was added and incubated on ice for 30 minutes. Samples were washed once with staining buffer prior to analysis by flow cytometry. As shown in FIG. 5, AG10048, AG10045, and AG10131 specifically bind to CD137, but not to any other tested family member or parent cell transfected with an empty vector.

Example 6
Ligand competition using ELISA and flow cytometry Antibodies were tested for their ability to block the binding of CD137 to their cognate ligand CD137L by both ELISA and flow cytometry assays. As shown in FIGS. 6a and 6b, all antibodies tested block the binding of CD137 to CD137L.

6a. Ligand Competitive Binding by ELISA Recombinant human CD137 (fused with human Fc and His tags) was diluted with PBS to 1 μg / mL and coated on Maxisorp plates overnight at 4 ° C. The plates were blocked with PBS supplemented with 3% skim milk for 1 hour at 37 ° C. After washing, a mixture of 50 μL of biotinylated CD137L (4 μg / mL) and test antibodies of various concentrations (8 1: 2 serial diluents in the range of 500 μg / mL to 2 μg / mL) totaling 100 uL is in each well. Was added to and incubated at 37 ° C. for 1 hour. The plates were washed 3 times, 100 μL of HRP conjugated neutravidin (1: 1000) was added to each well and incubated at 37 ° C. for 1 hour. The plates were washed as described above, 50 μL of TMB substrate solution was added, incubated at room temperature for 20 minutes, and then the reaction was stopped with 50 μL of H ₂ SO ₄ . As shown in FIG. 6a, all test antibodies AG10048, AG10045, and AG10131 block the binding of CD137 to CD137L. AG10131 shows the strongest or complete blocking capacity in the range of about uM, followed by AG10048, which shows significant blocking in the> uM range, followed by AG10059, which effectively blocks in the uM range. These data show that under the conditions tested, a wide variety of cross-reactive antibodies AG10131 and AG10058 are highly effective inhibitors of the interaction between CD137 and its ligand CD137L, depending on the reagents used. On the other hand, it is suggested that AG10059 shows only moderately effective blocking of the interaction between CD137 and its ligand CD137L. It should be noted that the reference antibody AC1097, which is cross-reactive with both human and monkey CD137, and AC1121, which reacts only with human CD137, show little blockade.

6b. Ligand Competitive Binding by Flow Cytometry The plasmid encoding full-length human CD137 was transiently expressed in HEK293F cells. Cells were washed with staining buffer (PBS supplemented with 1% BSA) and resuspended in staining buffer containing 100 nM test antibody. After incubating on ice for 30 minutes, 33 nM biotinylated CD137L was added to each well and incubated on ice for an additional hour. Cells were washed twice with staining buffer, 50 μL of staining buffer containing Alexa Fluor 647-conjugated streptavidin was added and incubated on ice for 30 minutes. Cells were washed once and analyzed by CytoFlex flow cytometry. As shown in FIG. 6b, all three antibodies tested can block the binding of CD137 and CD137L in a concentration-dependent manner. AG10131 shows the strongest blocking ability, followed by AG10048 with significant blocking, followed by AG10059 with less effective blocking. These data show that a wide variety of cross-reactive antibodies AG10131 and AG10024 are very effective in blocking the interaction between CD137 and its ligand CD137L, whereas AG10059 is between CD137 and its ligand CD137L. It suggests that it shows partial blockade of the interaction. In contrast, AC1097 reference antibodies that cross-react with both human and monkey CD137 show only partial blockade, whereas AC1121 reference antibodies that react only with human CD137 do not show blockade.

Example 7
Epitope Mapping A series of mutations (Table 5) were made in the extracellular domain of human CD137 to determine the binding region of the tested antibody at the amino acid residue level. These CD137 mutant plasmids were used to transfect HEK293F cells. Binding of the antibody to the human CD137 mutant was evaluated by flow cytometric analysis as previously described in Example 5 and is shown in FIG. 7A. The results, summarized in Table 5, show an interesting distinction that these antibodies show cross-reactivity with human, monkey, mouse, and rat CD137, as well as refined epitopes from hits from the Adagene library. AG10131 binds to all four species, while AG10048 binds to all three CD137 species, but not rat CD137. AG10048, AG10059, and AG10131 lose their ability to bind to the GFT34AAA, FSS53AAA, and FH92AA mutations, and their binding epitopes are within these regions, eg, amino acid residues 34-93 or 34-108 of SEQ ID NO: 1 (FIG. 1). See also 7B). AG10048 and AG10131 may bind to the same or very similar epitopes, and AG10059 may bind to epitopes different from AG10048 and AG10131.

The mutant construct meant distinguishing epitopes by AG10024, AG10059, and AG10131 from reference antibodies by AC1121 and AC1097. It is clear that the three antibodies AG10048, AG10059, and AG10131 all target very different epitopes from AC1121 and AC1097. AG10048, AG10059, and AG10131 differ from AC1121 in the region defined by the variants Hu_FH92AA and Hu_FSS53AAA and in some cases Hu_GTF34AAA, but AG10048, AG10059, and AG10131 are other than Hu_FH92AA and mouse, rat, and dog CD137. Their cross-reactivity with species is different, but differs from AC1097 in the region defined by most of the variants used, except for their cross-reactivity with monkeys. In some embodiments, AG10048, AG10059, and AG10131 or other antibodies disclosed herein do not bind to epitopes located within amino acid residues 115-156 of SEQ ID NO: 1. As also shown in FIG. 7A and Table 5, binding of human CD137 ligand to the wild-type pair mutant human CD137 was tested, with the observation that these antibodies block CD137 ligand binding to its receptor. It closely matches the antibody binding pattern.
Table 5 Epitope mapping

Example 8
Agonist activity of the antibody in the NFκB luciferase reporter assay Agonist activity of the antibody was evaluated using the NFκB reporter assay. 293T cells were transfected with a plasmid expressing human, monkey, or mouse CD137 along with an NFκB luciferase reporter plasmid. After 4 hours, 50 μL of cells were seeded into each assay well on a 96-well plate at a density of 0.4 × 10 ⁶ / mL. A total volume of 50 μL of antibody mixture containing the test antibody and a 3: 1 ratio cross-linked antibody (Fab'goat anti-human IgG FC) was added and incubated for 18 hours. After removing the medium, 50 μL of Passive Lysis Buffer (Promega E1980) was added and incubated at 37 ° C. for 30 minutes. 20 μL of lysate was transferred to a white plate and a luciferase substrate was added. The luminescence signals of fireflies and sea pansy were measured and their ratios were used for data analysis by GraphPad Prism 6.0 software. As shown in FIG. 8, all test antibodies activate the expression of the NFκB reporter gene when human and monkey CD137 is expressed as compared to the isotype control antibody. When mouse CD137 is expressed, AG10048 and AG10131, rather than AG10059, activate the expression of the NFκB reporter gene. This is consistent with previous observations that AG10048 and AG10131 bind to mouse CD137, whereas AG10059 does not.

Example 9
Agonist activity of the antibody in the T cell activation assay Agonist activity of the antibody was further confirmed in the T cell activation assay. 96-well cell culture plates in 1 x PBS with 50 μL of anti-CD3 antibody (2 μg / ml) alone or 50 μL of test antibody (60 μg / mL, 20 μg / mL, 6 μg / mL, 2 μg / mL, 0 μg / ml) mL) and coated overnight at 4 ° C. CD8 + T cells were isolated using a protocol according to the manufacturer's instructions. Cells were prepared at a density of 1 × 10 ⁷ cells / mL with RPMI1640 medium supplemented with 10% FBS. 200 μL of cells were seeded in each assay well and incubated at 37 ° C. in a 5% CO2 incubator for 4 days. Cells were checked under a microscope daily for proliferation. After 96 hours of incubation, 100 μL of supernatant was transferred to a new 96-well plate for IFN-γ detection. T cell proliferation was assayed using the Cell Titter Glow kit (Promega). As shown in FIG. 9, all antibodies tested compared to isotype control antibodies induced both CD8 + T cell proliferation and IFN-γ secretion in a dose-dependent manner.

Example 10
Antitumor activity in mouse syngeneic models Species cross-reactivity with mouse CD137 allows rapid in vivo functional assessment. AG10048 and AG10131 have been tested in multiple mouse syngeneic models. BALB / c mice (n = 8 per group) with 2 × 10 ⁶ H22 hepatocellular carcinoma cells (Xiao et. Al, Soluble PD-1 facilitates 4-1 BBL-liggered antibody immunity ageinst murine H22 hepatocellular carcinoma). 13 (6): 1823-30.), 5 × 10 ⁵ CT26 colon cancer cells, or 5 × 10 ⁵ EMT6 breast cancer cells were subcutaneously transplanted. When the tumor was established (> 50 mm ³ ), treatment with the isotype control antibody, AG10048, or AG10131 by intraperitoneal injection was initiated twice a week for up to 3 weeks. Tumor growth was monitored twice a week and mean tumor volume ± s. e. m. Reported as. As shown in FIGS. 10-13, both AG10058 and AG10131 showed strong in vivo antitumor activity in these different syngeneic mouse tumor models as compared to isotype control antibodies.

10a. CD137 agonist antibody shows antitumor effect in H22 mouse liver cancer model First, AG10058 or AG10131 was administered at a dose of 50 mg / kg twice a week for 3 weeks. Both molecules showed nearly 100% TGI (tumor growth inhibition) (FIG. 10, panel a). By immunohistochemical staining of CD4 and CD8 markers, AG10131 was found to be H22 tumor (Xiao et. Al, Solve PD-1 facilitates 4-1 BBL-triggered tumor immunohistochemical mechanism in vivo, hepatocellular carcinoma). 1823-30.) It has been shown to significantly increase the infiltration of CD4 + and CD8 + T cells in a microenvironment (Fig. 10, panel b). Further dose tapering up to 3 mg / kg showed approximately 100% TGI, suggesting that both molecules have potent antitumor activity (FIGS. 10, panels c and d). Further dose tapering of AG10131 to 1 mg / kg and 0.1 mg / kg also showed more than 50% TGI at 0.1 mg / kg and 1 mg / kg (FIG. 10, panel e).

10b. CD137 agonist antibody shows antitumor effect in CT26 mouse colon cancer model As shown in FIG. 10, CT26 mouse colon cancer model (Martinez-Forero et. Al, T cell costimulation with anti-CD137 monoclonal antibody 63 disease) Both AG10058 and AG10131 showed almost 100% TGI (tumor growth inhibition) at a dose of 50 mg / kg in polybicuitin-dependents signals. , Panel a). Further dose titration of AG10131 (FIG. 11, panel b) showed nearly 100% TGI at doses of 5 mg / kg and 1 mg / kg. At a dose of 0.1 mg / kg, about 40% TGI was achieved, demonstrating dose-dependent antitumor activity.

10c. EMT6 Breast Cancer Model Anti-tumor activity is further evaluated in an EMT6 mouse breast cancer syngeneic model (Shiand Siemann, Augmented antibody effects therapy by 4-1 BB antibody; 53) (Fig. 12). Both AG10048 and AG10131 showed about 100% inhibition of tumor growth.

10d. Mice that have completely responded to CD137 agonist antibody treatment remain tumor-free after re-challenge with new tumor cells.
After treatment with AG10048 or AG10131 in the CT26 tumor model for 3 weeks, mice with complete tumor regression were maintained for an additional month without treatment. Subsequently, mice maintained a complete response is the CT26 tumor cells 5 × 10 ⁵ on the opposite flank to day 62 re-challenge subcutaneously, were monitored for tumor growth. A re-challenge control group was simultaneously set up in naive mice inoculated with the same number of CT26 tumor cells. As shown in FIG. 13, treatment with AG10131 (1 and 5 mg / kg, see upper and lower panels of FIG. 13, respectively) showed strong antitumor activity in the CT26 tumor model and AG10131 (1 mg). 5/8 in the / kg group and 6/8 in the AG10131 (5 mg / kg group) showed a complete response for 60 days before re-challenge to CT26 tumor cells. Furthermore, these mice remained tumor-free after re-challenge with the same tumor cells, suggesting that specific antitumor memory developed in these mice.

To substantiate this hypothesis, spleen cells were collected from these tumor rejection re-challenge mice and control mice and co-cultured with mitomycin C growth-inhibited CT26 tumor cells in vitro for 7 days for tumor-specific memory. T cells were amplified. These splenocytes were then harvested and mixed with fluorescently labeled live CT26 tumor cells at different E / T ratios for 4 hours and tumor cell killing was detected by live / death staining and FACS analysis. As shown in FIG. 14, a marked increase in tumor cell killing was observed in splenocytes from tumor rejection re-challenge mice pretreated with both AG10048 and AG10131.

Example 11
AG10131-IgG4 does not induce ADCC effects Human CD8 ⁺ T cells were isolated from the peripheral blood of healthy donors by the EasySep human CD8 ⁺ T cell enrichment kit (StemCell Technologies) and PMA (50 ng / ml) + ionomycin (1 uM). ) Was stimulated in vitro for 18 hours. These activated CD8 ⁺ T cells were then labeled with Calcein-AM to function as target cells. NK cells from different healthy donors were isolated with a human NK isolation kit (StemCell Technologies) and allowed to function as effector cells. In the antibody-dependent cellular cytotoxicity (ADCC) assay, effector (NK) and target (activated CD8 ⁺ T) cells were cultured in a 96-well plate at a ratio of 5: 1 in the absence and presence of serially diluted antibodies. It was mixed for 4 hours under the conditions. The supernatant was then collected from each well and a fluorescent signal was detected on a plate reader SpectraMax i3x (Ex 488 nm, Em 520 nm). Isotype hIgG4 mAb was used as a negative control and humanized OKT3 (Novoprotein anti-CD3 hIgG1) was used as a positive control. The% dissolution was then calculated using the following formula: Dissolution% = [(experimental release) -mean (target + NK)] / [mean (target maximum) -mean (target alone)] x 100% (FIG. 15).

Example 12
Antibody Development Profiles Purified AG10048, AG10059, AG10131, and AC1097 were replaced with PB buffer (20 mM PB, 150 mM NaCl, pH 7.0) for feasibility evaluation. All experiments, including filtration, concentration and accelerated stress tests, were performed with PB buffer. A TSKgel column (Tosoh Bioscience G3000SWxl) was used for all SEC-HPLC analysis.

12a. Solubility All three antibodies can be concentrated above 100 mg / ml in PB buffer without overt precipitation (Table 6). The antibody was then adjusted to 20 mg / ml with PB buffer. Samples (10 μg each) were then analyzed by SEC-HPLC for detection of ultra high molecular weight (HMW) aggregates. As shown in the chromatogram (FIG. 16), no increase in HMW aggregates was observed at high concentrations (20 mg / ml) for all test antibodies.
Table 6 Antibody solubility

12b. Antibody stability under accelerated stress conditions Antibody stability was also investigated under accelerated stress conditions, and the results are summarized in Table 7. After 6 cycles of freezing (-80 ° C) and thawing (room temperature), all antibodies remain stable (FIG. 17). After 7 days at 50 ° C., there was little change in HMW aggregates or LMW fragments (Fig. 17). In long-term aging experiments (up to 28 days at 40 ° C.), all antibodies remained stable and there was no significant increase in HMW aggregates or LMW fragments (FIG. 17).
Table 7 Changes in HMW under acceleration conditions

Furthermore, the thermal stability measured by differential scanning calorimetry (DSC) indicates that both AG10131 and AG10024 are stable up to at least about 59 ° C. The midpoint of metastasis Tm (the characteristic temperature at which degenerative transition of almost all protein domains occurs) is shown in FIGS. 18 and 8 below.
Table 8 Thermal stability by DSC

Furthermore, the highest achieved concentrations of AG10131 and AG10048 after centrifugation were greater than 180 mg / mL and greater than 220 mg / mL, respectively.

Example 13
Safety profile of related species: mouse and cynomolgus monkey 13a. Repeated dose toxicity study of AG10131 in normal C57BL / 6 mice.
Repeated dose toxicity studies of AG10131 were performed in normal C57BL / 6 mice. Vehicle, AG10131 (100 mg / kg) on days 1, 4, 8 and 11 i. p. It was administered (10 mL / kg). Five female mice (7-8 weeks old) were included in each group. Mice were monitored daily for abnormal behavior and symptoms, and food intake and body weight were measured daily. On day 14, animals were euthanized for postmortem examination and other analysis. Blood is taken from each animal, two blood samples for each group for hematology (RBC, white blood cell, WBC, WBC percentage test), and the other three blood samples in the group for blood biochemistry (AL, AST, It was used for ALB, GLB, A / G, TBIL, ALP, GGT, and LDH) analysis. The following organs were collected from each mouse and stored in FFPE: heart, lung, thymus, liver, spleen, and kidney. FFPE blocks of liver tissue were prepared, sectioned and H & E stained for histopathological analysis.

There were no observations of abnormal behavior or unplanned animal deaths during the lifetime of all studies. Compared to vehicle treatment, AG10131 did not affect food intake and body weight. Postmortem examination showed no obvious lesions in mice in the treatment group with both AG10131. Hematological analysis showed no significant changes in blood biochemical parameters tested in mice treated with AG10131 (FIG. 19). No obvious abnormalities were found in the histopathological sections of the liver of all these mice (Fig. 20). Overall, AG10131 was well tolerated in this study and no significant toxicity was observed in mice.

13b. Repeated administration study of AG10131 in cynomolgus monkeys Repeated administration study of AG10131 was carried out in normal cynomolgus monkeys. Human IgG4 isotype controls (10 mg / kg), AG10131 (0.5 and 10 mg / kg) were added on days 0, 7, 14, and 22 i. v. It was administered (1 mL / kg). One male and one female cynomolgus monkey (3-5 years old) were included in each group. Animals were monitored daily for abnormal behavior and clinical signs, and food intake was measured daily. Body weight was measured on days (-15) and (-5) before administration, and on days 6, 13, 18, and 26 after administration. Hematology and hematological parameters are available on days (-12) and (-5) before administration, and on days 7, 14, 19, and 27 (10 mg / kg group only) after administration. ), And urinalysis was performed on days (-12) and (-5) before administration, and on days 6, 13, and 18 after administration. Animals in the 10 mg / kg group were euthanized on day 27 for postmortem examination and other analysis. Major organs were dissected and weighed. FFPE liver tissue blocks were prepared, sectioned and H & E stained for histopathological analysis.

During the lifetime of all studies, there were no observations of abnormal behavior or unplanned animal deaths in all groups. AG10131 treatment at 10 mg / kg did not affect food intake and body weight compared to vehicle treatment. There were no clinical signs, including reaction at the injection site. Post-mortem examination showed no obvious lesions or weight abnormalities in all organs examined in cynomolgus monkeys treated with 10 mg / kg AG10131. Hematology, hematology, and urine parameters are also within normal limits for all treatment groups (Fig. 21). Histopathological analysis of the liver showed no obvious abnormalities, including lymphocyte infiltration, after repeated doses of AG10131 at 10 mg / kg (FIG. 22). Overall, AG10131 was well tolerated in cynomolgus monkeys at weekly doses up to 10 mg / kg with no apparent toxicity detected.

Example 14
Pharmacokinetics of AG10131 in cynomolgus monkeys 14a. Pharmacokinetics of AG10131 in cynomolgus monkeys Pharmacokinetics studies of AG10131 were performed in naive cynomolgus monkeys. Three dose levels of AG10131 (10 mg / kg, 30 mg / kg, 100 mg / kg) were administered intravenously to three groups of monkeys. Each group includes 3 males and 3 females. Serum samples were 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, 336, 408, before and after administration. Collected at 504, 672, and 840 hours. The serum concentration of AG10131 was determined by ELISA.

AG10131 was rapidly eliminated on day 14 (336 hours) in 12 of 16 animals, i.e. all animals in the low and medium dose groups and 2 out of 6 animals in the high dose group. On day 21, two more animals from the high dose group showed rapid clearance. Serum concentrations in these 14 animals are either low or below the limit of quantification. This is consistent with the observation of anti-drug antibody production in these animals. Data from two animals that may not affect the pharmacokinetics of the high-dose group were fitted to predict pharmacokinetic parameters (FIG. 23). The half-life of AG10131 ranges from 7.3 to 8.8 days.

14b. Pharmacokinetics of AG10131 in Rats Pharmacokinetics studies of AG10131 were performed in naive SD rats. Three dose levels of AG10131 (10 mg / kg, 30 mg / kg, 100 mg / kg) were administered intravenous bolus to three groups of animals. Each group contains 15 males and 15 females. Serum samples were taken from 3 animals before and after administration 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168. , 240, 336, 408, 504, 672, and 840 hours at each time point. The serum concentration of AG10131 was determined by ELISA and the data were analyzed by Phoenix Professional V6.3.

Results: Low, medium and high dose PK parameters are similar (Fig. 24). The clearance rate of AG10131 is about 0.004 ml / kg / min. The half-life of AG10131 ranges from 11.5 to 14.6 days.

14c. Pharmacokinetics of AG10131 in Mice A pharmacokinetic study of AG10131 was performed in BALB / c mice approximately 8 weeks old. Three female BALB / c mice per administration group were intravenously injected with a test antibody containing AG10131 from the tail vein at 1 mg / kg. Blood samples (about 100 ul per sample) were collected 1 hour, 8 hours, 48 hours, 168 hours, and 336 hours after administration. Blank control blood was collected from 3 naive female mice without antibody administration. The serum concentration of each test antibody containing AG10131 was determined by ELISA and used for capture of anti-human IgG (Fc-specific) antibody and for detection of HRP-labeled anti-human IgG (Fab-specific) antibody.

All test antibodies, including isotype controls (AG10154), two benchmark antibodies (AC1020 and AC1021), and three Adamene antibodies (AG10131, AG10024, and AG10059), show comparable pharmacokinetics in mice (FIG. 25).

Example 15
Further Epitope Mapping Adopted a systematic approach to analyze epitopes at three levels of resolution: domain, motif, and residue to determine the binding epitope of the antibody presented herein by Adamene and other reference antibodies. did. The extracellular domain of CD137 containing four CRD motifs and four different species of CD137, such as human, monkey, mouse, and rat CD137 were used (Table 9). A series of human CD137 CRD motifs (cysteine-rich domains) and their constructs containing 1, 2, and 3 units of human CRD motifs (Table 9) are shown. Using a low copy number CEN / ARS-based vector, yeast S. Human CD137 CRD was expressed under the control of the inducible GAL1-10 promoter of S. cerevisiae (Boder and Wittrup (1997) Nat Biotechnology 15 (6): 535-7). Binding of the antibody to human CD137 CRD was evaluated by flow cytometric analysis and other techniques as previously described in Example 5 and is shown in FIG. 27.

The results are summarized in Table 9, where these antibodies selectively bind only to the CD137 target, but none of the non-CD137 targets listed in the table bind to humans, monkeys, mice, etc. by these antibodies. And species-specific cross-reactivity with CD137 from rat species is prominent, emphasizing sophisticated epitope coverage with a variety of hits screened from the Drugene Dynamic Precision Libraries. For example, AG10131 binds to CD137 in humans, monkeys, mice, rats, and dogs (not shown); AG10048 binds to CD137 in humans, monkeys, and mice, but not to rat CD137. On the other hand, AG10059 binds to both human and monkey CD137. In contrast, the reference antibody AC1121 from transgenic mice binds only to human CD137, while another reference antibody from the morphosis phage library binds to both human and monkey CD137. Note that for comparison, the human ligand CD137L interacts only with the human CD137 receptor, not the mouse CD137, and the mouse CD137L interacts only with the mouse CD137, not the human CD137 (see Table 9). ..
Table 9

As summarized in Table 9, in order to further analyze the binding motifs of these antibodies to the human CD137 target, the binding sites identified by Adage, other reference antibodies, were the dissected CD137 CRD motifs and combinations thereof. Sufficiently separated and noted as compared to CD137 ligand binding with: AG10048, AG10059, and AG10131 antibodies, as well as human CD137 ligand, are single CRDs or two CRD units (CRD2-) of human CD137. It does not bind to CRD3). The AG10048, AG10059, and AG10131 antibodies can bind to the three CRD units (CRD1-CRD2-CRD3) of human CD137, similar to the human CD137 ligand. The reference antibody AC1121 can also bind to the three CRD units of human CD137 (CRD1-CRD2-CRD3), which are the specific two CRD units of human CD137 (CRD1-CRD2). By comparison, three CRD units of human CD137 (CRD1-CRD2-CRD3) are required for binding by AG10048, AG10059, AG10131 antibody, and human CD137 ligand. Reference antibody AC1097 can bind to two CRD units (CRD3-CRD4), including three CRD units (CRD2-CRD3-CRD4) of human CD137. These show that Adagene's AG10048, AG10059, and AG10131 antibodies bind to epitopes covered by the three CRD units of human CD137 (CRD1-CRD2-CRD3) as well as human CD137 ligands. It is very different from the reference antibody AC1121 that binds to the two units of human CD137 (CRD1-CRD2) and the reference antibody AC1097 that binds to the two CRD units (CRD3-CRD4) of human CD137. In conclusion, the binding epitope of CD137 by the antibody of Adagene is very similar, if not duplicated, to the CD137 epitope by the human CD137L ligand, of the number of specific CRDs used and their CRD units required. Unlike the epitopes of the two reference antibodies (AC1121 with CRD1-CRD2 and AC1097 with CRD3-CRD4), as shown by the difference in points, the epitope between CD137-CD137L is as shown in FIG. , Recently reported by a crystal structure complex (Gilbreth, RN, Oganeseyan, V.Y., Amdoni, H., Novara, S., Grinberg, L., Barnes, A., Baca. , M. (2018) J. Biol. Chem. 293: 9880-9891).
Table 9B.

A series of mutations (Table 5) were made in the extracellular domain of human CD137 to determine the binding epitopes of the Adamene and reference antibodies at the amino acid residue level. These CD137 mutant plasmids were used to transfect HEK293F cells. Binding of the antibody to the human CD137 mutant was evaluated by flow cytometric analysis as previously described in Example 5 and is shown in FIG. 7A. The results, summarized in Table 5, show an interesting distinction that these antibodies show cross-reactivity with human, monkey, mouse, and rat CD137, as well as refined epitopes from hits from the Adagene library. AG10131 binds to human, monkey, mouse, and rat CD137, while AG10048 binds to human, monkey, and mouse CD137, but not rat CD137. Binding epitopes by AG10048, AG10059, and AG10131 are mapped to CRD1-CRD2-CRD3 units of CD137, which lose the ability to bind to the GFT34AAA, FSS53AAA, and FH92AA mutations, and their binding epitopes are within these regions. For example, it is shown to be at amino acid residues 34-93 or 34-108 of SEQ ID NO: 1 (see also FIG. 7B). AG10048 and AG10131 may bind to the same or very similar epitopes, and AG10059 may bind to epitopes different from AG10048 and AG10131. Single variants such as T35A, F36A, F53A, R66A, F72A, N83A, and F92A, along with binding by CD137L except R66A, which retains binding by its ligand to CD137, along with the Adamene antibodies AG10048, AG10059, and It shows the loss of binding to human CD137 by AG10131. However, the single mutants P32A and P49A lose the binding between CDL137L and CD137, but their effect on the interaction between the antibody and CD137 is altered. F125A indicates that AC1097 no longer binds to CD137, but does not affect binding by other antibodies, including human CD137L. In conclusion, the overall binding pattern by mutants throughout CD137 gives a clear message that the Adagene antibody and its reference antibody are distinguished in terms of binding site. The mutant construct meant distinguishing epitopes by AG10024, AG10059, and AG10131 from reference antibodies by AC1121 and AC1097. The three antibodies AG10048, AG10059, and AG10131 all target epitopes that are very different from AC1121 and AC1097. AG10048, AG10059, and AG10131 differ from AC1121 in the region defined by the variants Hu_FH92AA and Hu_FSS53AAA and in some cases Hu_GTF34AAA, but AG10048, AG10059, and AG10131 are other than Hu_FH92AA and mouse, rat, and dog CD137. Their cross-reactivity with species is different, but differs from AC1097 in the region defined by most of the variants used, except for their cross-reactivity with monkeys. In some embodiments, AG10048, AG10059, and AG10131 or other antibodies disclosed herein do not bind to epitopes located within amino acid residues 115-156 of SEQ ID NO: 1. As also shown in FIG. 7A and Table 5, binding of human CD137 ligand to the wild-type pair mutant human CD137 was tested, with the observation that these antibodies block CD137 ligand binding to its receptor. It closely matches the antibody binding pattern.

Example 16
Native CD137L signaling is blocked by AG10131 In vitro binding assay by ELISA demonstrated that AG10131 can block the interaction of recombinant CD137 with its ligand. To further functionally verify this ligand blocking activity of AG10131, a cellular NFκB luciferase reporter assay was performed. Briefly, 293T cells stably expressing the NFκB luciferase reporter were transfected with a DNA construct expressing human CD137, and the cells were co-cultured with human B-cell lymphoma cells Daudi or Razi in different proportions. The cell mixture was incubated overnight with a serial dilution of isotype control or ligand blocking anti-CD137 antibody and luciferase activity was measured using the Promega luciferase assay kit according to the manufacturer's instructions. Relative luciferase units (RLUs) were calculated relative to the level of luciferase expressed in 293T cells without antibody treatment.

As shown in FIG. 28, both Daudi (upper) and Razi (lower) cells expressed a functional CD137 ligand that activates the NFκB luciferase reporter in 293T cells. Addition of AG10131 to the co-culture system (right column) as compared to the isotype control antibody (left column) significantly inhibited NFκB signaling stimulated by both cell types, and the AG10131 antibody was found in Daudi and Razi. It suggests that it was able to functionally block CD137 signaling stimulated by the CD137 ligand expressed in both B lymphoma cells.

Example 17
Anti-CD137 antibody cross-linking in NFκB reporter assay Three anti-CD137 antibodies (AG10131, AC1121, and AC1097) were tested using a functional cellular NFκB reporter assay. As shown in FIG. 29, when cross-linked, all three anti-CD137 antibodies were able to stimulate human CD137 receptor signaling at comparable levels in a dose-dependent manner. The EC50 of the antibody-induced NFκB signaling activation response was in the same range for all three anti-CD137 antibodies. However, AC1121 showed unique properties different from AG10131 and AC1097. AC1121 was able to significantly activate human CD137 receptor signaling in the absence of cross-linking, but AG10131 and AC1097 were not. The EC50 of AC1121 with or without cross-linking in stimulation of CD137 receptor signaling was found to be at similar levels.

Example 18
AG10131 does not induce CDC The CDC activity of AG10131 was determined by direct binding of AG10131 to the purified C1q component of human complement by ELISA. As shown in FIG. 30, AG10131 and its human IgG4 isotype control antibody lack the ability to bind to the human complement C1q component in the concentration range tested, whereas the human IgG1 isotype control antibody can bind to C1q. This result suggests that AG10131 is also unlikely to induce complement-dependent cytotoxicity and is consistent with the IgG4 isotype framework.

Example 19
Anti-CD137 antibody AG10131 enhances tumor-invasive T lymphocytes In the in-vivo antitumor effect test in syngeneic mouse H22 liver cancer, EMT6 breast cancer, and CT26 colon cancer models shown in Example 10, AG10131 treatment strongly inhibited tumor growth. Demonstrated to do. Since AG10131 is an agonist antibody that activates T cells, treatment with AG10131 is expected to stimulate tumor-infiltrating T cells into the tumor microenvironment, thereby mediating antitumor effects in vivo. To evaluate the effect of AG10131 treatment on tumor-infiltrating lymphocytes, tumors from the in vivo antitumor effect test of AG10131 in mouse H22, EMT6, and CT26 cancer models were collected at the end of the study.

FIG. 31 shows representative IHC-stained images of mouse CD4 + and CD8 + T cells of H22 tumor (upper left), EMT6 tumor (upper right), and CT26 tumor (lower center). As shown in FIG. 32, T lymphocytes (either CD4 + or CD8 + T cells) were scarcely present in H22, EMT6, and CT26 tumors treated with vehicle controls, whereas AG10131 treatment treated CD4 + and CD8 + T cells. Significantly stimulated tumor infiltration (indicated by black arrows in FIG. 31). These data are consistent with the function of AG10131 as an immunoagonist by stimulating T cell proliferation, activation, and infiltration into the tumor microenvironment to mediate antitumor effects.

Example 20
Improvement of anti-tumor effect by combining anti-CD137 antibody AG10131 and anti-PD1 antibody in CT26 colon cancer model Next, the effect of combining anti-CD137 antibody AG10131 and anti-PD1 antibody was tested in CT26 colon cancer model. For tumor development, the CT26 tumor cells in the right lower abdomen region of each female BALB / c mice (3 × 10 ⁵⁾ were inoculated subcutaneously. When the average tumor volume reached 98 mm ³ , 10 mice were assigned to each experimental group. These groups include vehicle (PBS), 5 or 10 mg / kg AG10131, 10 mg / kg anti-PD-1, or a combination of 5 or 10 mg / kg AG10131 and 10 mg / kg anti-PD-mAb. It was administered by ip injection twice weekly for 3 weeks. Tumor volume was measured and each mouse was euthanized when the tumor reached an end volume of 2000 mm ³ or the final day (day 42), whichever came first.

As shown in FIG. 33, both AG10131 (5 mg / kg or 20 mg / kg) and anti-PD1 (10 mg / kg) slowed tumor progression, whereas AG10131 slowed tumor progression for a few more days, rarely tumors. Brought about a reduction in. However, almost all mice treated with either AG10131 or anti-PD1 eventually died of tumor progression. Importantly, when AG10131 (5 mg / kg or 20 mg / kg) was administered in combination with anti-PD1 (10 mg / kg), most mice essentially healed the tumor, with AG10131 (5 mg / kg) and anti-PD1. Only 2 (out of 10) or 1 (out of 10) of the combination of (10 mg / kg) or AG10131 (20 mg / kg) and anti-PD1 (10 mg / kg) avoided tumor suppression, respectively. These results demonstrate a strong synergistic effect of AG10131 and anti-PD1, suggesting that the combination of AG10131 and anti-PD1 may be effective in anti-PD1-resistant tumors.

Claims (57)

An isolated antibody that binds to the extracellular domain of human CD137, or an antigen-binding fragment thereof, which binds to one or more amino acid residues in amino acid residues 34-108 of SEQ ID NO: 1. Its antigen binding fragment. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment binds to one or more amino acid residues in amino acid residues 34 to 93 of SEQ ID NO: 1. 1 or claim, wherein the antibody or antigen binding fragment binds to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO: 1. Item 2. The antibody or antigen-binding fragment according to item 2. The antibody or antigen binding fragment binds to one or more of amino acid residues 34 to 36, one or more of 53 to 55, and one or more of 92 to 93 of SEQ ID NO: 1. The antibody or antigen-binding fragment according to claim 3. The antibody or antigen binding fragment does not bind to one or more of the amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. , The antibody or antigen-binding fragment according to any one of claims 1 to 4. The antibody or antigen-binding fragment according to claim 5, wherein the antibody or antigen-binding fragment does not bind to amino acid residues 109-112, 125, 126, 135-138, 150, and 151 of SEQ ID NO: 1. Any one of claims 1-6, wherein the antibody or antigen binding fragment is cross-reactive with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs. The antibody or antigen binding fragment according to the section. The antibody or antigen-binding fragment according to claim 7, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD137. The antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region.
The heavy chain variable region comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 711, HVR-H2 containing the amino acid sequence of SEQ ID NO: 735, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 759, and / or said. The light chain variable region comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: 783, HVR-L2 containing the amino acid sequence of SEQ ID NO: 807, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 831.
The antibody or antigen-binding fragment according to any one of claims 1 to 8. The antibody or antigen binding fragment of claim 9, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42. The antibody or antigen binding according to claim 10, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 617, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 618. Fragment. The antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region.
The heavy chain variable region comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 712, HVR-H2 containing the amino acid sequence of SEQ ID NO: 736, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 760, and / or said. The light chain variable region comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: 784, HVR-L2 containing the amino acid sequence of SEQ ID NO: 808, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 832.
The antibody or antigen-binding fragment according to any one of claims 1 to 8. The antibody or antigen binding fragment of claim 12, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62. 13. The antibody or antigen binding according to claim 13, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 619, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 620. Fragment. The antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region.
The heavy chain variable region comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 731, HVR-H2 containing the amino acid sequence of SEQ ID NO: 755, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 779, and / or said. The light chain variable region comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: 803, HVR-L2 containing the amino acid sequence of SEQ ID NO: 827, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 851.
The antibody or antigen-binding fragment according to any one of claims 1 to 8. The antibody or antigen-binding fragment of claim 15, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 72. The antibody or antigen binding according to claim 16, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 657, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 658. Fragment. An isolated antibody or antigen-binding fragment thereof that binds to the extracellular domain of human CD137, comprising a heavy chain variable region and a light chain variable region.
a) The heavy chain variable region includes HVR-H1, HVR-H2, and HVR-H3.
The HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of:
Formula (I): X ₁ TFX ₂ X ₃ YX ₄ IHWV (SEQ ID NO: 2), in the formula, X1 is F or Y, X2 is S or T, X3 is G, N, or S. And X4 are A, G, or W;
Formula (II): YSIX ₁ SGX ₂ X ₃ WX ₄ WI (SEQ ID NO: 3), in which X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is. A, D, G, N, S, or T; and formula (III): FSLSTX ₁ GVX ₂ VX ₃ WI (SEQ ID NO: 4), where X1 is G or S and X2 is A or G. And X3 is A, G, S, or T;
The HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of:
Formula (IV): LALIDDWX ₁ X ₂ DKX ₃ YSX ₄ SLKSRL (SEQ ID NO: 5), in the formula, X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y. And X4 is P or T;
Equation (V): IGX ₁ IYHSGX ₂ TYYX ₃ PSLKSRV (SEQ ID NO: 6), in which X1 is D or E, X2 is N or S, and X3 is N or S; and equation (VI). ): VSX ₁ ISGX ₂ GX ₃ X ₄ TYYADSVKGRF (SEQ ID NO: 7), in the formula, X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, X4 is S or T; and the HVR-H3 comprises the amino acid sequence according to formula (VII): ARX ₁ GX ₂ X ₃ X ₄ VX ₅ GDWFX ₆ Y (SEQ ID NO: 8) In the equation, X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T or V, and X5 is A, I, L, T. , Or V, and X6 is A, D, or G; and / or b) said light chain variable region comprising HVR-L1, HVR-L2, and HVR-L3.
The HVR-H3 comprises an amino acid sequence according to formula (VIII): X ₁ ASQX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ (SEQ ID NO: 9), where X1 is Q or R and X2. Is D, G, or S, X3 is I or V, X4 is G, R, S, or T, X5 is P, R, S, or T, and X6 is A, D, F, S, V, or Y, X7 is L or V, and X8 is A, G, or N;
The HVR-L2 comprises an amino acid sequence according to formula (IX): X ₁ ASX ₂ X ₃ X ₄ X ₅ GX ₆ (SEQ ID NO: 10), where X1 is A or D and X2 is N, S. , Or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and the HVR-L3 is: Includes amino acid sequences according to formulas selected from the group consisting of:
Formula (X): YCQQX ₁ YX ₂ X ₃ X ₄ T (SEQ ID NO: 11), in the formula, X1 is A, G, S, or Y, X2 is Q, S, or Y, and X3 is I. , L, T, or Y, and X4 is I, S, V, or W; and formula (XI): YCX ₁ QX ₂ X ₃ X ₄ X ₅ PX ₆ T (SEQ ID NO: 12), formula Among them, X1 is E or Q, X2 is P, S or Y, X3 is D, L, S, T or Y and X4 is D, E, H, S or T. , X5 is D, L, T, or W, and X6 is L, P, R, or V.
The antibody or its antigen-binding fragment. The HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 253 to 312, and the HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 313 to 372, said HVR-H3. Includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 373 to 432, the HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 433 to 492, and the HVR-L2 is the SEQ ID NO: The antibody or antigen-binding fragment of claim 18, comprising an amino acid sequence selected from the group consisting of 493 to 552, and said HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 553 to 612. .. The heavy chain variable region is SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53. , 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 , 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, and 131, and / or the light chain variable region comprises an amino acid sequence selected from the group. , SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 , 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 , 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, and 132. The antibody or antigen binding fragment of claim 18 or 19, comprising an amino acid sequence selected from the group. .. The HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of:
Formula (XII): X ₁ TFSX ₂ YWIHWV (SEQ ID NO: 853), in which X1 is F or Y and X2 is N or S;
Formula (XIII): YSIX ₁ SGX ₂ X ₃ WX ₄ WI (SEQ ID NO: 854), in which X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is. A, D, G, N, or S; and formula (XIV): FSLSTX ₁ GVX ₂ VX ₃ WI (SEQ ID NO: 855), in which X1 is G or S and X2 is A or G. , And X3 are A, G, or S;
The HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of:
Formula (IV): LALIWDX ₁ X ₂ DKX ₃ YSX ₄ SLKSRL (SEQ ID NO: 5), in the formula, X1 is A, D or Y, X2 is D or G and X3 is R, S or Y And X4 is P or T; and formula (XV): VSX ₁ ISGX ₂ GX ₃ X ₄ TYYADSVKGRF (SEQ ID NO: 856), where X1 is G, S, V, or Y and X2. Is A, D, S, or Y, X3 is D, G, or S, X4 is S or T; and the HVR-H3 comprises the amino acid sequence according to formula (VII): ARX _1. GX ₂ X ₃ X ₄ VX ₅ GDWFX ₆ Y (SEQ ID NO: 8), in the formula, X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T, Alternatively V, X5 is A, I, L, T, or V, and X6 is A, D, or G; and / or said HVR-L1 comprises an amino acid sequence according to formula (XVI). : X ₁ ASQX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ (SEQ ID NO: 857), in the formula, X1 is Q or R, X2 is D, G or S, X3 is I or V X4 is G, R, S, or T, X5 is P, R, S, or T, X6 is A, F, S, V, or Y, and X7 is L or V. Yes, and X8 is A or G;
The HVR-L2 comprises an amino acid sequence according to formula (XVII): X ₁ ASX ₂ X ₃ X ₄ X ₅ GX ₆ (SEQ ID NO: 858), in which X1 is A or D and X2 is N or S. X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and the HVR-L3 is of formula (XVIII). Including the amino acid sequence according to: YCQQX ₁ YX ₂ X ₃ WT (SEQ ID NO: 859), in the formula, X1 is A or G, X2 is S or Y, and X3 is I, L, or T.
The antibody or antigen-binding fragment according to claim 18. The HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 709 to 732, and the HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 733 to 756, said HVR-H3. Includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 757 to 780, the HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 781 to 804, and the HVR-L2 is the SEQ ID NO: The antibody or antigen-binding fragment according to claim 21, comprising an amino acid sequence selected from the group consisting of 805 to 828, and said HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 829 to 852. .. The heavy chain variable region is SEQ ID NO: 15, 17, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 61, 63, 65, 67, 71, 73, 75, 79. , 83, 85, and 87, and / or said light chain variable region comprises SEQ ID NOs: 16, 18, 32, 34, 36, 38, 40, 42, 44, 46. , 48, 50, 54, 62, 64, 66, 68, 72, 74, 76, 80, 84, 86, and 88, claim 21 or 22 comprising an amino acid sequence selected from the group consisting of Antibody or antigen binding fragment. The antibody comprises a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NOs: 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, It comprises an amino acid sequence selected from the group consisting of 645, 647, 649, 651, 653, 655, 657, and 659, and / or said light chain is SEQ ID NO: 614, 616, 618, 620, 622, 624. , 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, and 660, comprising an amino acid sequence selected from the group. Item 2. The antibody or antigen-binding fragment according to any one of Items 21 to 23. The HVR-H1 comprises the amino acid sequence of SEQ ID NO: 711 or 731, the HVR-H2 comprises the amino acid sequence of SEQ ID NO: 735 or 755, the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 759 or 779, said HVR. 21. The HVR-L2 comprises the amino acid sequence of SEQ ID NO: 783 or 803, the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 807 or 827, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 831 or 851. The antibody or antigen binding fragment of the description. The antibody or antigen-binding fragment of claim 25, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 or 71, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42 or 72. 26. The antibody of claim 26, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 617 or 657, and the light chain comprises the amino acid sequence of SEQ ID NO: 618 or 658. Antigen binding fragment. The HVR-H1 contains the amino acid sequence of SEQ ID NO: 712, the HVR-H2 contains the amino acid sequence of SEQ ID NO: 736, the HVR-H3 contains the amino acid sequence of SEQ ID NO: 760, and the HVR-L1 contains the amino acid sequence of SEQ ID NO: 784. 21. The antibody or antigen-binding fragment according to claim 21, wherein the HVR-L2 comprises the amino acid sequence of SEQ ID NO: 808 and the HVR-L3 comprises the amino acid sequence of SEQ ID NO: 832. 28. The antibody or antigen binding fragment of claim 28, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62. 29. The antibody or antigen binding fragment of claim 29, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 619, and the light chain comprises the amino acid sequence of SEQ ID NO: 620. The antibody or antigen binding fragment, as measured by surface plasmon resonance, binds to human CD137 with the following K _D 100 nM, antibody or antigen-binding fragment of any one of claims 1 to 30. The antibody or antigen-binding fragment, as measured by surface plasmon resonance, binds to human CD137 with the following K _D 50 nM, an antibody or antigen binding fragment of claim 31. Any one of claims 18-32, wherein the antibody or antigen binding fragment is cross-reactive with a CD137 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs. The antibody or antigen binding fragment according to the section. The antibody or antigen-binding fragment according to claim 33, wherein the antibody or antigen-binding fragment binds to cynomolgus monkey CD137. The antibody or antigen-binding fragment according to any one of claims 1 to 34, wherein contact with the antibody or antigen-binding fragment reduces the activity of human CD137 expressed in human cells. The antibody or antigen-binding fragment has a half-inhibition concentration (IC ₅₀ ) of about 100 nM or less for blocking the binding of human CD137 to human CD137L in vitro, according to any one of claims 1 to 34. The antibody or antigen binding fragment described. Claims 1-34, wherein the antibody or antigen-binding fragment completely blocks the binding of human CD137 to human CD137L in vitro when the antibody or antigen-binding fragment is provided at a concentration of about 1 μM or higher. The antibody or antigen-binding fragment according to any one of the following items. The antibody or antigen binding according to any one of claims 1 to 34, wherein the antibody or antigen binding fragment blocks one or more aspects of CD137 signaling stimulated by CD137L in cells expressing CD137. Fragment. The antibody or antigen-binding fragment according to any one of claims 1 to 34, wherein the activity of human CD137 expressed in human cells increases upon contact with the antibody or antigen-binding fragment. The antibody or antigen-binding fragment according to claim 39, wherein contacting a cell expressing CD137 with the antibody or antigen-binding fragment increases NF-κB-dependent transcription. The antibody or antigen-binding fragment according to any one of claims 1 to 40, wherein the antibody comprises a human IgG2 Fc region. The antibody or antigen-binding fragment according to any one of claims 1 to 41, wherein the antibody comprises a human IgG4 Fc region. The antibody or antigen-binding fragment of claim 42, wherein the human IgG4 Fc region comprises an S241P mutation, numbered Kabat. The heavy chain variable region includes SEQ ID NOs: 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 and 163, 165, 167, 169, 171 and 173. , 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 223 , 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, and 251 encoded by a polynucleotide comprising a sequence selected from the group and / or said. The light chain variable regions are SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, Claims 18-20 encoded by a polynucleotide comprising a sequence selected from the group consisting of 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, and 252. The antibody or antigen-binding fragment according to any one of the above. The heavy chains are SEQ ID NOs: 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703. , 705, and / or the light chain of the light chain is encoded by a polynucleotide comprising a sequence selected from the group consisting of, 705, and 707, and / or said light chain of SEQ ID NO: 662, 664, 666, 668, 670, 672, 674, 676, 678, 24. Claim 24, encoded by a polynucleotide comprising a sequence selected from the group consisting of 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, and 708. The antibody or antigen binding fragment according to. A polynucleotide encoding an antibody or antigen-binding fragment according to any one of claims 1 to 45. A polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 133-252. A vector containing the polynucleotide according to any one of claims 46 to 47. The vector according to claim 48, wherein the vector is an expression vector. A host cell comprising the vector according to claim 48 or 49. A method for producing an antibody or antigen-binding fragment, which comprises culturing the host cell according to claim 50 under conditions suitable for producing the antibody or antigen-binding fragment. 51. The method of claim 51, further comprising recovering the antibody or antigen binding fragment produced by the cells. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 45 and a pharmaceutically acceptable carrier. A method of treating a subject's cancer in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1-45. Method. 54. The method of claim 54, further comprising administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. 55. The method of claim 55, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapies, radiotherapy, and chemotherapy. The at least one additional therapeutic agent is pomalist, levrimid, renalide, pomalide, salidamide, DNA alkylated platinum-containing derivative, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD. -L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR Antibodies, cetuximab, rituximab, trussumab, penbrolizumab, radiotherapy, single irradiation, fractionated irradiation, local irradiation, whole organ irradiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adopted The method of claim 55 or 56, selected from the group consisting of T cells, anti-cancer vaccines, and tumor-dissolving viruses.